Distinct cerebellar engrams in short-term and long-term motor learning

Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF–PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF–PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF–PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF–PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF–PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF–PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.Image stabilization in the visual field via the vestibulo-ocular reflex and optokinetic response requires accurate extraocular muscle synergies that rely on long-term plastic calibrations in the cerebellar flocculus (FL) and its downstream target vestibular nuclei (VN) (1–8). Long-term depression (LTD) in parallel fiber-Purkinje cell (PF–PC) synapses has been postulated as a possible mechanism for this plastic calibration based on many lines of mutant mice that lack both LTD and learning (9–12). However, LTD’s role in motor learning has been recently questioned by a few mutant mice lines (13) and mice with pharmacological treatments (14) that showed lack of LTD but no impairment of learning. Furthermore, long-term potentiation in PF–PC synapses has been also shown to be involved in the motor learning (15). Recent evidence indicates that various forms of synaptic plasticity works synergistically and can compensate each other when one is missing in cerebellar motor learning (16). Despite the apparently contradictory results, no direct evidence for the decrease or increase of synaptic AMPA receptors (AMPARs) has been shown in physiological motor learning. To elucidate in vivo neuronal substrates for motor learning in wild-type mouse, we examined individual PF–PC synapses using quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL) (17) combined with morphometric EM analysis after adaptation of horizontal optokinetic response (HOKR). HOKR is a simple model of cerebellar motor learning and the FL, which is a phylogenetically preserved cerebellar lobule, is involved in the adaptation of HOKR (4, 18). We found that LTD as a form of AMPARs decrease does occur in PF–PC synapses after 1-h training of HOKR. However, LTD lasted less than 24 h, and instead we discovered a drastic elimination of these synapses, which gradually emerged over 5 d of HOKR training. These results indicate distinct engrams in the cerebellum for short-term and long-term memory in motor learning.     

Modeling memory consolidation during posttraining periods in cerebellovestibular learning

Long-term depression (LTD) at parallel fiber–Purkinje cell (PF–PC) synapses is thought to underlie memory formation in cerebellar motor learning. Recent experimental results, however, suggest that multiple plasticity mechanisms in the cerebellar cortex and cerebellar/vestibular nuclei participate in memory formation. To examine this possibility, we formulated a simple model of the cerebellum with a minimal number of components based on its known anatomy and physiology, implementing both LTD and long-term potentiation (LTP) at PF–PC synapses and mossy fiber–vestibular nuclear neuron (MF–VN) synapses. With this model, we conducted a simulation study of the gain adaptation of optokinetic response (OKR) eye movement. Our model reproduced several important aspects of previously reported experimental results in wild-type and cerebellum-related gene-manipulated mice. First, each 1-h training led to the formation of short-term memory of learned OKR gain at PF–PC synapses, which diminished throughout the day. Second, daily repetition of the training gradually formed long-term memory that was maintained for days at MF–VN synapses. We reproduced such memory formation under various learning conditions. Third, long-term memory formation occurred after training but not during training, indicating that the memory consolidation occurred during posttraining periods. Fourth, spaced training outperformed massed training in long-term memory formation. Finally, we reproduced OKR gain changes consistent with the changes in the vestibuloocular reflex (VOR) previously reported in some gene-manipulated mice.Long-term depression (LTD) at parallel fiber–Purkinje cell (PF–PC) synapses in the cerebellar cortex has been thought to be the major mechanism of motor learning (1). This Marr–Albus–Ito hypothesis (2, 3), however, has been challenged since Miles and Lisberger’s proposal (4) that long-term potentiation (LTP) at mossy fiber–vestibular nuclear neuron (MF–VN) synapses, not LTD at PF–PC synapses, underlies vestibuloocular reflex (VOR) gain adaptation (4–7). In a recent study on optokinetic response (OKR) gain adaptation, we found evidence that might resolve the controversy: LTD at PF–PC synapses (PF-LTD) and LTP at MF–VN synapses (MF-LTP) play different roles in OKR adaptation (8–10). Namely, PF-LTD accounts for short-term memory in PCs during 1-h training, whereas MF-LTP forms long-term memory in VN after the 1-h training that accumulates during repeated trials of 1-h training. It thus appears as if short-term memory formed in PCs during 1-h training is transferred to VN after training to consolidate as long-term memory (8–10).To investigate the mechanisms of this memory transfer and posttraining consolidation, we conducted a computer simulation study using a simple theoretical model of the cerebellovestibular system including both LTD and LTP at PF–PC synapses and MF–VN synapses. Although several theoretical models have addressed the question of how multiple plasticity mechanisms work together in cerebellar motor learning (11–14), none has explicitly taken the memory consolidation process during posttraining periods into consideration. Our model reproduced previously reported oculomotor behavioral data obtained from wild-type mice (8–10). Notably, the simulated nuclear long-term memory formed mostly after training and relatively little during training, consistent with the hypothesis of posttraining memory consolidation. We also conducted computer simulation of OKR gain adaptation in three strains of gene-manipulated mice in which either PF-LTD or PF-LTP was impaired specifically, or inhibition of PCs was blocked, and reproduced gain changes consistent with those in VOR gain adaptation in those mice (15–17).     

Progressive maturation of silent synapses governs the duration of a critical period

During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95–dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.Immature cortical neural networks, which are formed primarily under genetic control (1), require experience and training to shape and optimize their functional properties. This experience-dependent refinement is considered to be a general developmental process for all functional cortical domains and typically peaks during their respective critical periods (CPs) (2, 3). Known examples for CPs span functional domains as diverse as filial imprinting and courtship song learning in birds (4, 5); cognitive functions, such as linguistic or musical skills in humans (6, 7); and likely best studied, the different features of sensory modalities (3). CPs are characterized by the absolute requirement for experience in a restricted time window for neural network optimization. Lack of visual experience during the CP for visual cortex refinements can, for example, cause irreversible visual impairment (8). Refinements during the CP play an essential role (9). Although some functions can be substantially ameliorated after the CP, they are rarely optimally restored.It is believed that the neural network refinement is based on synapse stabilization and elimination (10–12) and includes forms of long-term synaptic plasticity to remodel excitatory synapses of principal neurons (13, 14). Although long-term plasticity at these excitatory synapses is instructive for shaping neural networks for functional output and their expression coincides with CPs, it is not known whether the remodeling itself governs the duration of CPs. In contrast, only permissive mechanisms have been shown to terminate CPs. Among these, the developmental increase of local inhibition appears to be the dominating mechanism to regulate cortical plasticity and CPs (15–17). Additionally, extracellular matrix remodeling is involved, as well as receptors of immune signaling, such as paired Ig-like receptor B (PirB), or axon pathfinding, such as Nogo (18–21). However, a specific function to directly regulate synapse remodeling during initial neural network optimization is not known and a potential instructive function of PirB was described for adult cortical plasticity but not plasticity of the initial synapse remodeling during CPs (22).AMPA receptor-silent synapses have been proposed to be efficient plasticity substrates during early cortical network refinements (13, 23, 24). Silent synapses are thought to be immature, still-developing excitatory synapses containing only NMDA receptors (NMDARs) but lacking AMPA receptors (AMPARs) (23, 24). They are functionally dormant but can evolve into fully transmitting synapses by experience-dependent insertion of AMPARs, a plasticity process thought to occur frequently in developing cortices (10). Although they appear as the ideal synaptic substrate for CP plasticity and their maturation correlates with sensory experience (10, 25), it has not been experimentally tested whether maturation of silent synapses indeed causes the termination of critical periods. This conceptual model contrasts with the current view that increased local inhibition and the expression of plasticity brakes ends critical periods (18–20, 26). We hypothesize that experience-dependent unsilencing of silent synapses, which results in strengthening and maturation of excitatory synapses, governs network stabilization and refinement during critical periods, and that the progressive decrease of silent synapses leads to the closure of critical periods.Experience-dependent cortical plasticity is classically tested with ocular dominance (OD) plasticity (ODP) in the primary visual cortex (V1), induced by monocular deprivation (MD). In the binocular region of mouse V1, neurons respond to sensory inputs from both eyes, but activity is dominated by afferents from the contralateral eye. During the critical period, a brief MD induces an OD shift of visually evoked responses in V1 toward the open eye (27–29). This juvenile ODP is mediated by a reduction of deprived eye responses in V1 and is temporally confined to a critical period (30, 31).A molecular candidate regulating the cellular basis of critical period plasticity is postsynaptic density protein-95 (PSD-95), whose expression in the visual cortex increases on eye opening and thus the onset of visual experience (32). PSD-95 promotes the maturation of AMPA receptor-silent excitatory synapses in hippocampal neurons and is required for activity-driven synapse stabilization (33–35). In juvenile PSD-95 KO mice, ODP displays the same features as in WT mice (36). However, as adult PSD-95 KO mice have not yet been analyzed, it is unknown whether PSD-95 is essential for the closure of critical periods. Thus, PSD-95 appeared to be the ideal molecular candidate to test our conceptual model that progressive silent synapse maturation marks the closure of critical periods.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.     

Distinct kinetics of synaptic structural plasticity,memory formation,and memory decay in massed and spaced learning

Long-lasting memories are formed when the stimulus is temporally distributed (spacing effect). However, the synaptic mechanisms underlying this robust phenomenon and the precise time course of the synaptic modifications that occur during learning remain unclear. Here we examined the adaptation of horizontal optokinetic response in mice that underwent 1 h of massed and spaced training at varying intervals. Despite similar acquisition by all training protocols, 1 h of spacing produced the highest memory retention at 24 h, which lasted for 1 mo. The distinct kinetics of memory are strongly correlated with the reduction of floccular parallel fiber–Purkinje cell synapses but not with AMPA receptor (AMPAR) number and synapse size. After the spaced training, we observed 25%, 23%, and 12% reduction in AMPAR density, synapse size, and synapse number, respectively. Four hours after the spaced training, half of the synapses and Purkinje cell spines had been eliminated, whereas AMPAR density and synapse size were recovered in remaining synapses. Surprisingly, massed training also produced long-term memory and halving of synapses; however, this occurred slowly over days, and the memory lasted for only 1 wk. This distinct kinetics of structural plasticity may serve as a basis for unique temporal profiles in the formation and decay of memory with or without intervals.During learning, memories are formed in a specific population of neuronal circuits and are consolidated for persistence (1, 2). These memory processes are supported by discrete subcellular events such as reversible modifications in the efficacy of synaptic transmission (3–5) or persistent structural modifications in the size and number of synaptic connections (6–8). However, how these synaptic modifications relate to the dynamics of formation and decay of memories in behaving animals remains elusive. Memory formation and its persistence are also sensitive to the temporal features of stimulus presentation, as observed in the well-known “spacing effect.” Training trials that include resting intervals between them (spaced training) produce stronger and longer-lasting memories than do the same number of trials with no intervals (massed training) (9). The spacing effect has been observed in a variety of explicit and implicit memory tasks (10–13), and the molecular mechanisms supporting this phenomenon have been reported (14–18). Various intracellular signaling molecules such as CREB (19), mitogen-activated protein (MAP) kinase (20, 21), and PKA (22, 23) underlie the spacing effect and are implicated in the remodeling of neuronal structures (23). In vitro studies showed that spaced stimuli induced the protrusion of new filopodia (20) and the recruitment of new synapses (24) in hippocampal neurons. However, despite the existence of numerous behavioral and molecular studies, no conjoint study has elucidated the synaptic correlates that underpin the expression of the spacing effect during learning. Here we studied the temporal evolution and decay of memory and its correlation with synaptic modifications during learning with distinct temporal patterns of training.We used an adaptation of the horizontal optokinetic response (HOKR), which is a simple model of cerebellum-dependent motor learning. It is a compensatory eye movement for stabilization of the visual image on the retina during horizontal motion of the surroundings. A surrounding that oscillates horizontally at a given frequency causes retinal slips in naive animals and facilitates HOKR 1 h after training (HOKR adaptation) (25–27). The amount of adaptation can be quantitatively monitored, and the flocculus (Fl), which is a phylogenetically preserved cerebellar lobule, is involved in the adaptation of the HOKR (28, 29). These features render this paradigm as an experimental model, useful for investigating neural correlates and mechanisms involved in motor learning. In a previous study, we showed that the short-term adaptation of HOKR induced by 1-h training was accompanied by a rapid and transient reduction (28%) in the number of AMPA receptors (AMPARs) in parallel fiber (PF) to Purkinje cell (PC) synapses, whereas the long-term adaptation induced by repeated 1-h training over 5 d was accompanied by a slowly developing reduction (45%) of PF–PC synapses (30). Despite recent controversies on the role of long-term depression (LTD) and a postulated role of long-term potentiation in cerebellar motor learning (31–33), this study first showed that LTD as a form of reduced AMPARs in PF–PC synapses does occur in physiological learning.In the present study, we further examined how the spacing effect is correlated with the structural plasticity in PF–PC synapses. We showed that spaced training including 1-h intervals induced stable long-lasting memories within 4 h after the training, which was accompanied by a rapid and long-lasting (>1 mo) reduction of PF–PC synapses after a transient reduction in AMPAR density and shrinkage of PF–PC synapses and PC spines. One hour of massed training also induced a gradual reduction of the PF–PC synapses, which reached the same level as that observed for the spaced training 5 d later but recovered faster within 10 d. The time course corresponded well with the slower establishment and quicker decay of long-lasting memory induced by massed training. The tight correlation observed between the structural modifications and the kinetics of long-lasting memory pinpoints the distinct temporal regulation of synaptic connections as a mechanism underlying the spacing effect.     

Sphingolipid metabolism governs Purkinje cell patterned degeneration in Atxn1[82Q]/+ mice

Patterned degeneration of Purkinje cells (PCs) can be observed in a wide range of neuropathologies, but mechanisms behind nonrandom cerebellar neurodegeneration remain unclear. Sphingolipid metabolism dyshomeostasis typically leads to PC neurodegeneration; hence, we questioned whether local sphingolipid balance underlies regional sensitivity to pathological insults. Here, we investigated the regional compartmentalization of sphingolipids and their related enzymes in the cerebellar cortex in healthy and pathological conditions. Analysis in wild-type animals revealed higher sphingosine kinase 1 (Sphk1) levels in the flocculonodular cerebellum, while sphingosine-1-phosphate (S1P) levels were higher in the anterior cerebellum. Next, we investigated a model for spinocerebellar ataxia type 1 (SCA1) driven by the transgenic expression of the expanded Ataxin 1 protein with 82 glutamine (82Q), exhibiting severe PC degeneration in the anterior cerebellum while the flocculonodular region is preserved. In Atxn1[82Q]/+ mice, we found that levels of Sphk1 and Sphk2 were region-specific decreased and S1P levels increased, particularly in the anterior cerebellum. To determine if there is a causal link between sphingolipid levels and neurodegeneration, we deleted the Sphk1 gene in Atxn1[82Q]/+ mice. Analysis of Atxn1[82Q]/+; Sphk1^−/− mice confirmed a neuroprotective effect, rescuing a subset of PCs in the anterior cerebellum, in domains reminiscent of the modules defined by AldolaseC expression. Finally, we showed that Sphk1 deletion acts on the ATXN1[82Q] protein expression and prevents PC degeneration. Taken together, our results demonstrate that there are regional differences in sphingolipid metabolism and that this metabolism is directly involved in PC degeneration in Atxn1[82Q]/+ mice.

Purkinje cells (PCs) integrate all afferent sensorimotor information using their massive dendritic tree and form the exclusive output source of the cerebellar cortex. Based on the highly structured and repetitive organization of the cerebellar cortex, PCs are often regarded as a homogeneous population of neurons. However, for several decades anatomical and immunohistochemical studies have emphasized that different subtypes of PCs can be identified in the cerebellar cortex, including transverse zones (e.g., the anterior vs. flocculonodular cerebellum), parasagittal modules (e.g., AldolaseC [AldoC]-positive vs. AldoC-negative domains), and others (such as apex vs. sulcus or vermis vs. hemispheres) (reviewed in ref. 1). Regional differences in PC density (2), dendritic arborization (3), morphology of organelles (4), and axonal thickness (5) can be observed across the cerebellar cortex. The most commonly studied pattern is defined by AldoC (also known as ZebrinII) expression, which divides PC subtypes in parasagittal bands with distinct molecular footprints (6–9) and distinct electrophysiological intrinsic and synaptic properties (10–12), as well as input/output connectivity (13, 14). Cerebellar heterogeneity is also illustrated by the nonhomogeneous and nonrandom PC degeneration across the cerebellar cortex observed in a broad spectrum of cerebellar pathologies. These cerebellar pathologies, displayed by patients or rodent models, can result from genetic mutations, such as leaner (15), Harlequin (16), sticky (17), Slc9a6^−/− (18), and Cav2.1^−/− (19), as well as brain ischemia (20), viral infections (21), prion diseases (22), and alcohol abuse, such as alcohol-related cerebellar degeneration (23). As described by Sarna and Hawkes (24), not all patterned neurodegenerations are similar. Two main types of patterned degeneration have been observed: 1) pathologies in which the flocculonodular cerebellum is more resistant, leading to a clear separation regarding the progression of the neurodegeneration between anterior cerebellum and flocculonodular lobule, and 2) pathologies in which PC degeneration affects primarily AldoC-negative domains, leading to parasagittal compartmentalization of neurodegeneration. Thus far, mechanisms explaining the patterned neurodegeneration of PCs have not been revealed, although some cellular pathways have been investigated (25), or suggested, such as the metabolism of sphingolipids (24). Sphingolipids are essential bioactive sphingoid-based complex lipids that regulate physiological processes such as cell differentiation, neurogenesis, synaptic strengthening, and cell survival (26–29). Particularly, sphingosine-1-phosphate (S1P) is a potent bioactive modulator, acting intracellularly or in an autocrine/paracrine manner (30). S1P is the product of sphingosine (Sph) phosphorylation, which is catalyzed by sphingosine kinases (Sphks; Sphk1 and Sphk2) and can be reversed by dephosphorylation through sphingosine phosphate phosphatases (Sgpps; Sgpp1 and Sgpp2) or irreversibly degraded by S1P lyase (31). Both Sph and S1P are part of ceramide metabolism, known to be a central mediator in programmed cell death (32). The S1P/ceramide ratio, or rheostat, is decisive for cell fate in several tissues (33), and tight regional control has been correlated with local neuroprotective effects in structures of the central as well as peripheral nervous system (34, 35). PCs are known to be sensitive to sphingolipid levels, as decreasing ceramide and Sph levels by blocking the de novo ceramide synthesis pathway through the serine palmitoyltransferase inhibitor led to a decrease in survival rate of PCs in culture (36). Conversely, enrichment of culture media with ceramide, Sph, or dihydroceramide promoted PC survival. In vivo, PC degeneration was observed in mutant models targeting proteins involved in the sphingolipid pathway, such as ceramide synthase CerS1 (37), Acid Ceramidase 3 (38), and Saposin D (39, 40). Typically, all those mutants show either preferential degeneration of the anterior cerebellum or AldoC-negative PCs, patterns which could appear similar but remain clearly distinct after proper scrutinization. In mouse models of Niemann–Pick disease types A and C, featuring loss of function of acid ceramidase or Niemann–Pick C1 Protein, respectively, patterned PC degeneration was clearly described (41). Although most of these data indicate an antiapoptotic role of S1P in PCs, accumulation of long-chain bases Sph and S1P has also been reported as proapoptotic in vitro (42) and in vivo (43, 44), and restoring long-chain base levels rescued the neurodegenerative phenotype in the CerS1 mouse model (45). Altogether, this stresses the impact of sphingolipid homeostasis on PC fate. Therefore, we investigated the possible role of local sphingolipid metabolism on cerebellar patterned neurodegeneration. Here, we show that in adult wild-type mice, Sphk1 levels are higher in the flocculonodular region, while the levels of its product, S1P, are higher in the anterior region. We then investigated changes in sphingolipid levels in the Atxn1[82Q]/+ mice model of the spinocerebellar ataxia type 1 (SCA1) (46). The neurodegeneration observed in this model results from the expression and localization of the polyglutamine mutant protein in the nucleus (47). In Atxn1[82Q]/+ mice, degeneration primarily affects the anterior region, while the flocculonodular lobule remains spared over an extended course of the disease (46, 48–50). We found that this prevalence correlates with increased S1P levels and region-specific decreases in Sphk1 and Sphk2 expression levels compared to wild-type mice. To test the potential contribution of Sphk1 activity to neurodegeneration, we crossed Atxn1[82Q]/+ mice with Sphk1 knockout mice (Sphk1^−/−). Whereas Sphk1^−/− mice did not exhibit cerebellar defects or significant changes in S1P levels, the deletion of Sphk1 in Atxn1[82Q]/+ mice resulted in a neuroprotective effect on a subset of PCs. The deletion of Sphk1 affects the expression of ATXN1[82Q] protein and reduces somatic PC atrophy in defined domains reminiscent of AldoC-positive PC modules. Taken together, we show that sphingolipid metabolism can impact the expression of misfolded proteins and, thereby, play a major role in patterned PC degeneration.     

Three small vesicular pools in sequence govern synaptic response dynamics during action potential trains

During prolonged trains of presynaptic action potentials (APs), synaptic release reaches a stable level that reflects the speed of replenishment of the readily releasable pool (RRP). Determining the size and filling dynamics of vesicular pools upstream of the RRP has been hampered by a lack of precision of synaptic output measurements during trains. Using the recent technique of tracking vesicular release in single active zone synapses, we now developed a method that allows the sizes of the RRP and upstream pools to be followed in time. We find that the RRP is fed by a small-sized pool containing approximately one to four vesicles per docking site at rest. This upstream pool is significantly depleted by short AP trains, and reaches a steady, depleted state for trains of >10 APs. We conclude that a small, highly dynamic vesicular pool upstream of the RRP potently controls synaptic strength during sustained stimulation.

Synaptic depression is primarily due to the exhaustion of a finite pool of synaptic vesicles (SVs), the readily releasable pool (RRP) (1). In several preparations, it has been suggested that the RRP is not homogeneous (2–4). At the calyx of Held synapse, the response to presynaptic depolarization originates from two pools of equivalent sizes, the slowly releasing pool (SRP) and the fast releasing pool (FRP) (5); the FRP has been further split into primed FRP and superprimed FRP (6–9). Likewise, in cerebellar mossy fiber synapses (10–12) and in hippocampal mossy fiber synapses (13), two subpools resembling the FRP and SRP of the calyx of Held have been identified. In each of these two types of synapses, recent studies using total internal reflection fluorescence microscopy have indicated the presence of several subpools of SVs within 100 nm from the active zone (AZ) membrane (14, 15). The organization of these various pools and their roles during sustained or repeated synaptic stimulations have been extensively examined but remain uncertain (review: refs. 16–19). In particular, it remains unclear whether the SRP, the primed FRP, and the superprimed FRP release their SVs according to a parallel scheme, to a sequential scheme, or to a mixed parallel-sequential scheme (5, 8, 12, 20–25), even though recent studies often favor sequential schemes (9, 26, 27). In certain additional synapses [cerebellar parallel fiber to molecular layer interneuron (28, 29), molecular layer interneuron to molecular layer interneuron (30), and hippocampal mossy fiber to interneuron (31)], studies carried out at the level of individual AZs (“simple synapse recording”: ref. 32) suggest a sequential binding of incoming SVs to a “replacement site” and an associated “docking site” before release. These results suggest two sequentially arranged pools of up to one docked SV and one replacement SV per release site. Altogether, it is plausible that central synapses involve a common basic scheme where two to three subpools constitute the RRP or are closely associated with the RRP. However, the exact structure of these pools and their respective roles during synaptic activity remain to be established.The standard method to distinguish RRP-related subpools has been to measure the kinetics of SV release during sustained presynaptic depolarization (5, 7, 11–13, 20). However, recent studies have suggested that the pattern of responses to individual action potentials (APs) in a train also gives information on the originating subpools of the released SVs (9, 28). In addition, several lines of evidence indicate that modifications of RRP-related subpools are accompanied by changes in release kinetics during a train. For instance, the well-known shift from synchronous to asynchronous release that accompanies synaptic depression (33) has been proposed to be linked to RRP depletion (34–36). Furthermore, it has been shown that after each AP in a train, the latency distribution of synchronous release is biphasic, with a fast component reflecting the release of docked SVs and a slow component reflecting an uninterrupted sequence of docking followed by release of SVs initially located in replacement sites (“two-step release”: ref. 29). As the proportion of fast vs. slow release changes markedly during a train, it provides information on the dynamics of docked and replacement vesicular pools. Altogether, a study of the pattern of release as a function of AP number, together with a quantitative assessment of release kinetics during trains, may provide decisive information on RRP-related subpools.In the present work, we take advantage of simple synapse recordings at cerebellar parallel fiber–molecular layer interneuron (PF–MLI) synapses, together with modeling, to investigate changes in RRP-related subpools during AP trains. Our results indicate the existence of an unexpectedly small-sized pool (intermediate pool; 1.2 to 4 SVs per release site) located upstream of replacement SVs. We propose that in this preparation, three pools of comparable sizes (intermediate, replacement, and docked SVs) are placed sequentially to guide SVs toward exocytosis.     

Territories of heterologous inputs onto Purkinje cell dendrites are segregated by mGluR1-dependent parallel fiber synapse elimination

In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.Monoinnervation of cerebellar Purkinje cells (PCs) by single climbing fibers (CFs) is established in the early postnatal period (1–3). The soma of a neonatal PC is innervated by more than five CFs with similar synaptic strengths, from which a single CF is functionally strengthened (4, 5). The strengthened (“winner”) CF starts dendritic translocation, whereas the other weaker (“loser”) CFs remaining on the soma are eliminated (6–8). In this process, P/Q-type voltage-dependent Ca²⁺ channels (VDCCs) promote functional differentiation and dendritic translocation of winner CFs, and the early phase of CF synapse elimination (9–11), whereas the late phase of CF synapse elimination is critically dependent on the formation of parallel fiber (PF) synapses and activation of the type 1 metabotropic glutamate receptor (mGluR1)-protein kinase Cγ (PKCγ) pathway (12–17).Segregated dendritic innervation by CFs and PFs is another distinguished feature of the PC synaptic wiring. Although hundreds of thousands of PFs innervate the distal dendritic domain, a single CF monopolizes the proximal dendritic domain, and both innervate a narrow intermediate domain (18). Given that both dendritic translocation of winner CFs and formation of PF synapses proceed upwards from the base of the dendritic tree (6, 19), CFs and PFs must compete with each other to establish their segregated territories. However, the developmental route and the underlying mechanisms of this process are unknown.Our findings indicate that CF and PF territories on PC dendrites are dynamically refined during the early postnatal period, and that the mGluR1-PKCγ signaling pathway regulates segregation by promoting PF synapse elimination. Thus, this signaling cascade plays key roles in sculpting the excitatory synaptic wiring in PCs by eliminating both redundant CF synapses from the soma (3, 20) and competing PF synapses from the proximal dendrites.     

Social experiences switch states of memory engrams through regulating hippocampal Rac1 activity

In pathological or artificial conditions, memory can be formed as silenced engrams that are unavailable for retrieval by presenting conditioned stimuli but can be artificially switched into the latent state so that natural recall is allowed. However, it remains unclear whether such different states of engrams bear any physiological significance and can be switched through physiological mechanisms. Here, we show that an acute social reward experience switches the silent memory engram into the latent state. Conversely, an acute social stress causes transient forgetting via turning a latent memory engram into a silent state. Such emotion-driven bidirectional switching between latent and silent states of engrams is mediated through regulation of Rac1 activity–dependent reversible forgetting in the hippocampus, as stress-activated Rac1 suppresses retrieval, while reward recovers silenced memory under amnesia by inhibiting Rac1. Thus, data presented reveal hippocampal Rac1 activity as the basis for emotion-mediated switching between latent and silent engrams to achieve emotion-driven behavioral flexibility.

Animals are required to flexibly retrieve memories according to their emotional states for achieving optimal behaviors in an ever-changing environment. To understand the underlying mechanisms of such regulation, extensive effort has been devoted to studying the impact of emotion on memory processes (1–5). For instance, stress can block memory retrieval through hormones, neuroinflammation, or depression of synapses in both human and animal models (6–12), while reward and novelty can facilitate both the formation and maintenance of memories (13, 14). However, how emotion might directly impact the memory engram remains elusive. The proposed theory and experimental demonstrations have revealed the presence of multiple states of memory engrams, such as silent, latent, and active states (15–17). In the silent state, only artificial activation of engram cells is capable of inducing memory expression, whereas latent engram cells can be activated by a natural conditioned stimulus to drive the engram into the active state for memory retrieval. It is intriguing to note that training either mouse models of Alzheimer’s disease or protein synthesis-inhibited mice are reported to yield only silent engrams, and such silent engrams could be turned into a latent state (18–22) through artificial manipulations of either optical stimulation–induced long-term potentiation (LTP) or virus-driven overexpression of activated PAK1. However, the physiological significance of the different states of engrams, particularly the silent engram, remains unclear. In the course of studying the functions of reversible forgetting (23, 24), we became interested in testing the idea that the emotional impact on memory retrieval could be mediated through switching the engram between latent and silent states, while reversible forgetting may play a role in making such switching.To investigate this idea, we tested the effects of acute social reward (SR) and social stress (SS) on memory retrieval. Since mating and fighting are widely perceived and used as behavioral stimuli for evoking feelings of reward and stress, respectively (25–27), we adopted a modified short procedure for evoking acute emotion through social interactions. For SR treatment, a single experimental male mouse is exposed to two females brought from different home cages for 10 min. Such a subtle positive experience is sufficient to enhance retrieval of contextual fear memory (28). For SS treatment, a single experimental male mouse is exposed to a group of five male littermates for 10 min. This is a hostile social environment in which the experimental mouse fights with other littermates during this time window. Such a subtle stressful experience is sufficient to significantly reduce 24-h contextual fear memory (28). Based on these two paradigms of acute social experiences, we investigated how emotion affects memory retrieval through alterations in engram states.     

T-type channel blockade impairs long-term potentiation at the parallel fiber–Purkinje cell synapse and cerebellar learning

Ca_V3.1 T-type channels are abundant at the cerebellar synapse between parallel fibers and Purkinje cells where they contribute to synaptic depolarization. So far, no specific physiological function has been attributed to these channels neither as charge carriers nor more specifically as Ca²⁺ carriers. Here we analyze their incidence on synaptic plasticity, motor behavior, and cerebellar motor learning, comparing WT animals and mice where T-type channel function has been abolished either by gene deletion or by acute pharmacological blockade. At the cellular level, we show that Ca_V3.1 channels are required for long-term potentiation at parallel fiber–Purkinje cell synapses. Moreover, basal simple spike discharge of the Purkinje cell in KO mice is modified. Acute or chronic T-type current blockade results in impaired motor performance in particular when a good body balance is required. Because motor behavior integrates reflexes and past memories of learned behavior, this suggests impaired learning. Indeed, subjecting the KO mice to a vestibulo-ocular reflex phase reversal test reveals impaired cerebellum-dependent motor learning. These data identify a role of low-voltage activated calcium channels in synaptic plasticity and establish a role for Ca_V3.1 channels in cerebellar learning.Neurotransmission at the parallel fiber (PF) and Purkinje cell (PC) synapse plays a pivotal role in cerebellar motor learning probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, postsynaptic Ca²⁺ signaling at PF–PC spines may not be dominated by ionotropic glutamatergic receptors, as postsynaptic N-methyl-D-aspartate receptors (NMDARs) are not prominently present at this site and AMPA receptors are predominantly impermeable for calcium ions (4, 5). PCs bear different voltage-dependent Ca channels including P/Q-type (6–8) and T-type channels (9, 10). The spines of PCs contain a high density of Ca_V3.1 T-type channels (11), which can be readily activated by typical bursts of PF activity that occur during sensory stimulation (12–14). To date, the function of the PF to PC synapse plays a pivotal role in cerebellar motor learning, probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, T-type channels during PF–PC plasticity induction and cerebellar learning has not been explored.In cerebellar PCs, the elevation of Ca²⁺ in the spine has been suggested to control directly the sign of the changes in synaptic weights (15). Long-term depression (LTD) induction requires conjunctive stimulation of the climbing fibers (CFs) and PFs, which triggers a large supralinear calcium entry mediated by mGluR1, inositol triphosphate (IP3) receptors and voltage-gated calcium channels (16–19). In contrast, long-term potentiation (LTP) develops after PF stimulation only and requires a moderate [Ca²⁺]_i elevation (15). Here, we evaluated the hypothesis that Ca_V3.1 T-type channel activation is essential for LTP and LTP-dependent motor learning.We first looked at PF–PC plasticity of T-type channel blockade/deletion, and then investigated both in vitro and in vivo the dynamics of PC activity as well as the motor behavior of both wild-type and Ca_V3.1 KO mice. Because, in our experiments, motor behavior appears to be impaired in tests requiring a refined body balance, we have analyzed vestibulo-ocular reflex (VOR) adaptation, a learning paradigm more specifically dependent on vestibulo-cerebellar function. We show all three processes to be impaired after T-type channel functional inactivation. We propose that T-type calcium channels contribute to the definition of the learning rules in the cerebellar cortex.     

Sleep promotes the formation of dendritic filopodia and spines near learning-inactive existing spines

Changes in synaptic connections are believed to underlie long-term memory storage. Previous studies have suggested that sleep is important for synapse formation after learning, but how sleep is involved in the process of synapse formation remains unclear. To address this question, we used transcranial two-photon microscopy to investigate the effect of postlearning sleep on the location of newly formed dendritic filopodia and spines of layer 5 pyramidal neurons in the primary motor cortex of adolescent mice. We found that newly formed filopodia and spines were partially clustered with existing spines along individual dendritic segments 24 h after motor training. Notably, posttraining sleep was critical for promoting the formation of dendritic filopodia and spines clustered with existing spines within 8 h. A fraction of these filopodia was converted into new spines and contributed to clustered spine formation 24 h after motor training. This sleep-dependent spine formation via filopodia was different from retraining-induced new spine formation, which emerged from dendritic shafts without prior presence of filopodia. Furthermore, sleep-dependent new filopodia and spines tended to be formed away from existing spines that were active at the time of motor training. Taken together, these findings reveal a role of postlearning sleep in regulating the number and location of new synapses via promoting filopodial formation.

Learning and memory consolidation are associated with the rewiring of neuronal network connectivity (1–3). Previous studies have shown that motor training leads to the formation and elimination of postsynaptic dendritic spines of pyramidal neurons in the primary motor cortex (M1) (4–8). Learning-induced new spines stabilize and persist over long periods of time (4). The extent of spine remodeling correlates with behavioral improvement after learning (4, 9), and the disruption of spine remodeling impairs learned motor behavior (10–12). These studies suggest that learning-induced new synapses contribute to changes in neuronal circuits that are likely important for the retention of learned behaviors (13, 14).Cumulative evidence suggests that sleep affects synaptic structural plasticity in many brain regions (15–17). For example, sleep has been shown to promote spine formation and elimination in developing somatosensory and visual cortices (18, 19). In the motor cortex, sleep promotes branch-specific formation of new dendritic spines following motor learning and selectively stabilizes learning-induced new synaptic connections (11, 12). Sleep has also been shown to regulate dendritic spine numbers in hippocampal CA1 area (20–22). In addition, many lines of evidence have revealed the function of sleep in increasing, decreasing, or stabilizing synaptic strength and neuronal firing in various brain regions (23–31). Together, these studies strongly suggest that sleep has an important role in promoting synaptic structural plasticity in neuronal circuits during development and after learning.While sleep promotes the formation of new spines after learning (12), it remains unknown how postlearning sleep regulates new synapse formation along dendritic branches. Synapse formation is a prolonged process often involving the generation of dendritic filopodia, thin and long protrusions without bulbous heads (32–35). These highly dynamic filopodia have been shown to initiate the contact with presynaptic axonal terminals and transform into new spines (36, 37). It is not known whether sleep promotes new spine formation via filopodia formation and subsequent transformation. Furthermore, it is also unclear whether sleep-dependent formation of new dendritic protrusions (filopodia and spines) is distributed on dendritic branches in a random or nonrandom manner. On the one hand, new synapses may be formed in clusters with synapses of similar functions to allow nonlinear summation of inputs important for increasing memory storage capacity (9, 38–43). On the other hand, new connections may be formed preferentially near less active/strong synapses to avoid competition for limited synaptic resources (44–47).In this study, we found that dendritic filopodia and spines formed after motor training were partially clustered with existing spines on apical tuft dendrites of layer 5 (L5) pyramidal neurons in the mouse primary motor cortex. Posttraining sleep was critical for the clustered formation of new filopodia, some of which were transformed into new spines. In addition, the clustered new filopodia and spines tended to be formed near existing spines that were inactive at the time of motor training. These findings reveal a role for sleep in neuronal circuit plasticity by promoting clustered spine formation via dendritic filopodia near learning-inactive existing spines.     

Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in the hippocampal CA3 circuit

Homeostatic plasticity of intrinsic excitability goes hand in hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging, and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2 to 3 d induces an up-regulation of both synaptic transmission at CA3–CA3 connections and intrinsic excitability of CA3 pyramidal neurons. Intrinsic plasticity was found to be mediated by a reduction of Kv1.1 channel density at the axon initial segment. In activity-deprived circuits, CA3–CA3 synapses were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a reduction in presynaptic Kv1.1 function. Further support for the down-regulation of axonal Kv1.1 channels in activity-deprived neurons was the broadening of action potentials measured in the axon. We conclude that regulation of the axonal Kv1.1 channel constitutes a major mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

Chronic modulation of activity regimes in neuronal circuits induces homeostatic plasticity. This implicates a regulation of both intrinsic excitability (homeostatic plasticity of intrinsic excitability) and synaptic transmission (homeostatic plasticity of synaptic transmission) to maintain network activity within physiological bounds (1). In most cases, these two forms of homeostatic plasticity act synergistically but involve different molecular actors. Homeostatic intrinsic plasticity is associated with the regulation of voltage-gated ion channels (2–9), while homeostatic synaptic plasticity involves the regulation of postsynaptic receptors to neurotransmitters (10–17) or the regulation of the readily releasable pool of synaptic vesicles (18–20). However, the function of voltage-gated ion channels is not limited to the control of intrinsic excitability. Several studies point to the role of axonal voltage-gated channels in shaping presynaptic action potential (AP) waveform and subsequently controlling neurotransmitter release and synaptic transmission (21–35). Moreover, some studies describe homeostatic plasticity of the AP waveform via voltage-gated channel regulation (36–38), while other studies report an absence of this phenomenon (39).Kv1.1 channels are responsible for the fast-activating, slow-inactivating D-type current (I_D) in CA3 neurons. This current has been shown to create a delay in the onset of the first AP and to determine intrinsic excitability in various neuronal types, including CA1 and CA3 pyramidal neurons of the hippocampus (3, 40), L5 pyramidal neurons of the cortex (26, 34), and L2/3 fast-spiking interneurons of the somatosensory cortex (41, 42). Furthermore, Kv1.1 channels have been shown to control axonal AP width and subsequently presynaptic calcium entry and neurotransmitter release. In fact, pharmacological blockade of Kv1.1 channels broadens presynaptic APs and increases synaptic transmission at neocortical and hippocampal glutamatergic synapses and at cerebellar GABAergic synapses (21, 22, 26, 30, 32, 41, 43, 44). Moreover, Kv1.1 channels have been shown to be responsible for the phenomenon of depolarization-induced analog digital facilitation of synaptic transmission (d-ADF). In fact, at CA3–CA3 and L5–L5 synapses, a somatic subthreshold depolarization of the presynaptic cell leads to inactivation of axonal Kv1.1 channels, inducing the broadening of the presynaptic AP, an increase in spike-evoked calcium entry, and a facilitation of presynaptic glutamate release (26, 31, 33, 34, 45, 46). Therefore, Kv1.1 channels control both intrinsic excitability and glutamate release in CA3 pyramidal neurons.Kv1.1 channels have been shown to be involved in homeostatic regulation of neuronal excitability. Chronic activity enhancement by kainate application leads to an increase in I_D current and a decrease in excitability in dentate gyrus granule cells (47). Conversely, chronic sensory deprivation leads to Kv1.1 channel down-regulation and enhancement of excitability in the avian cochlear nucleus (48). In this study, we examined whether the increase in synaptic transmission could also be due to Kv1.1 channel down-regulation, which would possibly explain the observed synergy between homeostatic plasticity of excitability and synaptic transmission.We show here that chronic activity deprivation induced with an antagonist of ionotropic glutamate receptors (kynurenate) in hippocampal organotypic cultures provokes both an increase in CA3 pyramidal cells excitability and an enhancement of synaptic transmission at monosynaptically connected CA3 neurons. Deprived cultures display a decrease in Kv1.1 channel staining in the axon initial segment. Bath application of dendrotoxin-K (DTX-K), a selective blocker of Kv1.1 channels, leads to a larger excitability increase in control cultures than in deprived cultures. Focal puffing of DTX-K on the axon increases excitability in control but not in deprived cultures, showing that homeostatic plasticity of excitability in deprived cultures is partly due to the down-regulation of axonal Kv1.1 channels. In addition, we found that axonal Kv1.1 down-regulation in deprived cultures is responsible for a spike broadening in CA3 neurons, leading to elevated release probability at CA3–CA3 synapses. Consistent with these observations, d-ADF, a Kv1.1-dependent form of synaptic facilitation, is present in control cultures but not in deprived cultures. Altogether, these results show that chronic activity blockade of the hippocampal CA3 circuit induces the down-regulation of axonal Kv1.1 channels leading to a homeostatic increase in both excitability and presynaptic release probability.     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.     

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors

GABA_A-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABA_A receptors, and requires a newly described interaction with GABARAP, a GABA_A receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.Interneurons that release γ-aminobutyric acid (GABA) are a major source of inhibitory signaling in vertebrate nervous systems, and play important roles in cognition, mood, and behavior (1, 2). Many of these inhibitory interneurons release GABA, which binds to ionotropic ligand-gated GABA_A receptors located at GABAergic synapses and at extrasynaptic sites, and these GABA_A receptors are sites of action for benzodiazepine and barbiturates (3). GABA_A receptors are dynamic, with continuous exchange between synaptic and extrasynaptic sites in the plane of the membrane, as well as endocytic trafficking between the cell surface and intracellular compartments (3–6). GABA_A receptor cell surface expression is believed to be required for formation of GABAergic synapses based on studies with heterogeneously-expressed GABA_A receptors (7). However, the role of GABA_A receptors in preserving GABAergic synapses has not yet been described in a native neuronal environment.GABAergic synapses localize to both the axon initial segment (AIS) as well as somatodendritic sites of target neurons (2, 8, 9). In the cerebellum, basket and stellar interneurons project specific axon terminals to the AISs of Purkinje cells, forming GABAergic “pinceau” synapses (10). Formation of these pinceau synapses depends on a steep gradient of the cell adhesion molecule neurofascin, which is enriched at the AIS (11, 12). Both GABAergic pinceau synapses and the neurofascin gradient are missing in mice with cerebellar knockout out of the membrane adaptor ankyrin-G (11, 13). Ankyrin-G coordinates multiple proteins at AISs including voltage-gated sodium channels (VGSC), KCNQ2/3 channels, 186-kDa neurofascin, and beta-4 spectrin (14). A role of ankyrin-G in stabilizing GABAergic synapses outside of the the AIS of cerebellar neurons has not been explored.Assembly of AISs as well as their GABAergic synapses requires giant ankyrin-G, which contains a 7.8-kb alternatively spliced nervous system-specific exon found only in vertebrates (14). In addition to ANK repeats and a beta-spectrin-binding domain, giant ankyrin-G (480-kDa ankyrin-G) contains 2,600 residues configured as an extended fibrous polypeptide (14–17). Giant ankyrin-G has been assumed to be confined to AISs and nodes of Ranvier and a general role for ankyrin-G in GABAergic synapse stability at other cellular sites has not been entertained (14, 15, 18).Here we report that giant ankyrin-G is present in extrasynaptic microdomains on the somatodendritic surfaces of hippocampal and cortical neurons, and describe a giant ankyrin-G–based mechanism required for cell surface expression of GABA_A receptors and for maintaining somatodendritic GABAergic synapses. We find that somatodendritic giant ankyrin-G inhibits GABA_A receptor endocytosis through an interaction with the GABA_A receptor-associated protein (GABARAP). This previously unidentified role for giant ankyrin-G provides a newly resolved step in the formation of GABA_A-receptor-mediated circuitry in the cerebral cortex as well as a rationale for recent linkage of human mutations in the giant ankyrin exon with autism and severe cognitive dysfunction (19).     

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.The postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2–5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6–10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14–18). These MAGUK proteins share conserved modular structures consisting of three PDZ domains (19, 20) and one SH3-GK supermodule (21). PDZ domains of MAGUKs bind to a conserved motif at the extreme C-terminal region of GluN2 subunits of NMDARs (16, 22). PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23–25). Single-particle tracking of AMPARs provides evidence that AMPAR/Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27, 28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30–32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).PSD-95 and PSD-93 include N-terminal palmitoylation sites that enable PSD-95 and PSD-93 to associate with membrane lipids. N-terminal palmitoylation of PSD-95 is necessary for its synaptic localization, clustering of receptors (33–35), and stability at the PSD (36). PSD-95 palmitoylation regulates synaptic strength by controlling the accumulation of AMPARs at the PSD (35). Consistent with these results, a recent immunogold electron microscopy (immuno-EM) mapping of the positions of the two ends of the PSD-95 molecule at the PSD shows that its N terminus is located at the membrane, whereas its C terminus is farther away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3, 4, 37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites. SAP97 contains a L27 domain at the N terminus (31, 38), which might be involved in self-association, and has a role in sorting and trafficking of AMPARs and NMDARs (39) but is not required for basal synaptic transmission (40).The MAGUK family proteins interact with a host of other proteins in the PSD, such as GKAP (41, 42), which binds to the GK domain of the MAGUKs, whereas GKAPs in turn bind Shank and Homer (43–45). Both Shank and Homer can interact with actin-associated proteins, thus indirectly linking the core PSD structure to the actin system prevalent in the cytoplasm of dendritic spines (45). MAGUKs interact with signaling complexes such as AKAPs (46, 47), K channels (48), and postsynaptic adhesion molecules such as neuroligin (49, 50). With an average density of 300–400 molecules per PSD (51, 52), the MAGUKs outnumber glutamate receptors by a significant margin. With so many potential binding partners, the MAGUKs are positioned to play an important role in organizing glutamate receptors as well as other scaffolding and signaling molecules at the PSD (53).We have examined the consequences of knocking down three key MAGUKs on excitatory synaptic transmission and found an ∼80% reduction in both AMPAR and NMDAR synaptic responses (54). Interestingly, despite the rather ubiquitous distribution of MAGUKs at excitatory synapses, the reduction in synaptic AMPAR-mediated transmission appeared to be attributable primarily to an all-or-none loss of functional synapses. We present evidence that after the knockdown, there is an initial uniform decrease in AMPARs across all synapses, but over a 4-d period, a consolidation process in which a “winner-take-all” phenomenon occurs (54).Here, we have used EM tomography (3, 4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3, 4, 37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure. Here, we show that knocking down three key synaptic MAGUKs results in a profound loss of vertical filaments and the electron-dense materials manifested by the PSD. The loss of MAGUKs is accompanied by a dramatic loss of both NMDAR- and AMPAR-type structures at the PSD.     

miR-182 targeting reprograms tumor-associated macrophages and limits breast cancer progression

The protumor roles of alternatively activated (M2) tumor-associated macrophages (TAMs) have been well established, and macrophage reprogramming is an important therapeutic goal. However, the mechanisms of TAM polarization remain incompletely understood, and effective strategies for macrophage targeting are lacking. Here, we show that miR-182 in macrophages mediates tumor-induced M2 polarization and can be targeted for therapeutic macrophage reprogramming. Constitutive miR-182 knockout in host mice and conditional knockout in macrophages impair M2-like TAMs and breast tumor development. Targeted depletion of macrophages in mice blocks the effect of miR-182 deficiency in tumor progression while reconstitution of miR-182-expressing macrophages promotes tumor growth. Mechanistically, cancer cells induce miR-182 expression in macrophages by TGFβ signaling, and miR-182 directly suppresses TLR4, leading to NFκb inactivation and M2 polarization of TAMs. Importantly, therapeutic delivery of antagomiR-182 with cationized mannan-modified extracellular vesicles effectively targets macrophages, leading to miR-182 inhibition, macrophage reprogramming, and tumor suppression in multiple breast cancer models of mice. Overall, our findings reveal a crucial TGFβ/miR-182/TLR4 axis for TAM polarization and provide rationale for RNA-based therapeutics of TAM targeting in cancer.

It is well known that the nonmalignant stromal components in tumors play pivotal roles in tumor progression and therapeutic responses (1, 2). Macrophages are a major component of tumor microenvironment and display considerable phenotypic plasticity in their effects toward tumor progression (3–5). Classically activated (M1) macrophages often exert direct tumor cytotoxic effects or induce antitumor immune responses by helping present tumor-related antigens (6, 7). In contrast, tumoral cues can polarize macrophages toward alternative activation with immunosuppressive M2 properties (6–8). Numerous studies have firmly established the protumor effects of M2-like tumor-associated macrophages (TAMs) and the association of TAMs with poor prognosis of human cancer (9–11). However, how tumors induce the coordinated molecular and phenotypic changes in TAMs for M2 polarization remains incompletely understood, impeding the designing of TAM-targeting strategies for cancer intervention. In addition, drug delivery also represents a hurdle for therapeutic macrophage reprogramming.Noncoding RNAs, including microRNAs, have been shown to play vital roles in various pathological processes of cancer (12). The microRNA miR-182 has been implicated in various developmental processes and disease conditions (13–15). Particularly, it receives extensive attention in cancer studies. Prevalent chromosomal amplification of miR-182 locus and up-regulation of its expression in tumors have been observed in numerous cancer types including breast cancer, gastric cancer, lung adenocarcinoma, colorectal adenocarcinoma, ovarian carcinoma, and melanoma (16–21). miR-182 expression is also linked to higher risk of metastasis and shorter survival of patients (20, 22–24). Functional studies showed that miR-182 expression in cancer cells plays vital roles in various aspects of cancer malignancy, including tumor proliferation (25–29), migration (30, 31), invasion (16, 32, 33), epithelial-mesenchymal transition (34–36), metastasis (21, 37, 38), stemness (30, 39, 40), and therapy resistance (41, 42). A number of target genes, including FOXO1/3 (18, 21, 43–45), CYLD (46), CADM1 (47), BRCA1 (27, 48), MTSS1 (34), PDK4 (49), and SMAD7 (35), were reported to be suppressed by miR-182 in cancer cells. Our previous work also proved that tumoral miR-182 regulates lipogenesis in lung adenocarcinoma and promotes metastasis of breast cancer (34, 35, 49). Although miR-182 was established as an important regulator of cancer cell malignancy, previous studies were limited, with analyses of miR-182 in cultured cancer cells and transplanted tumors. Thus, the consequences of miR-182 regulation in physiologically relevant tumor models, such as genetically modified mice, have not been shown. More importantly, whether miR-182 also plays a role in tumor microenvironmental cell components is unknown.In this study, we show that miR-182 expression in macrophages can be induced by breast cancer cells and regulates TAM polarization in various tumor models of mice. In addition, miR-182 inhibition with TAM-targeting exosomes demonstrates promising efficacy for cancer treatment.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.