Metabotropic glutamate receptor signaling is required for NMDA receptor-dependent ocular dominance plasticity and LTD in visual cortex

A feature of early postnatal neocortical development is a transient peak in signaling via metabotropic glutamate receptor 5 (mGluR5). In visual cortex, this change coincides with increased sensitivity of excitatory synapses to monocular deprivation (MD). However, loss of visual responsiveness after MD occurs via mechanisms revealed by the study of long-term depression (LTD) of synaptic transmission, which in layer 4 is induced by acute activation of NMDA receptors (NMDARs) rather than mGluR5. Here we report that chronic postnatal down-regulation of mGluR5 signaling produces coordinated impairments in both NMDAR-dependent LTD in vitro and ocular dominance plasticity in vivo. The data suggest that ongoing mGluR5 signaling during a critical period of postnatal development establishes the biochemical conditions that are permissive for activity-dependent sculpting of excitatory synapses via the mechanism of NMDAR-dependent LTD.Temporary monocular deprivation (MD) sets in motion synaptic changes in visual cortex that result in impaired vision through the deprived eye. The primary cause of visual impairment is depression of excitatory thalamocortical synaptic transmission in layer 4 of visual cortex (1–3). The study of long-term depression (LTD) of synapses, elicited in vitro by electrical or chemical stimulation, has revealed many of the mechanisms involved in deprived-eye depression (4). In slices of visual cortex, LTD in layer 4 is induced by NMDA receptor (NMDAR) activation and expressed by posttranslational modification and internalization of AMPA receptors (AMPARs) (5, 6). MD induces identical NMDAR-dependent changes in AMPARs, and synaptic depression induced by deprivation in vivo occludes LTD in visual cortex ex vivo (6–8). Manipulations of NMDARs and AMPAR trafficking that interfere with LTD also prevent the effects of MD (7, 9–11).Although NMDAR-dependent LTD is widely expressed in the brain (12, 13), it is now understood that different circuits use different mechanisms for long-term homosynaptic depression (14). For example, in the CA1 region of hippocampus, synaptic activation of either NMDARs or metabotropic glutamate receptor 5 (mGluR5) induces LTD. In both cases, depression is expressed postsynaptically as a reduction in AMPARs, but these forms of LTD are not mutually occluding and have distinct signaling requirements (15). A defining feature of mGluR5-dependent postsynaptic LTD in CA1 is a requirement for the immediate translation of synaptic mRNAs (16). In visual cortex, there is evidence that induction of LTD in layers 2–4 requires NMDAR activation, whereas induction of LTD in layer 6 requires activation of mGluR5 (17, 18).The hypothesis that mGluRs, in addition to NMDARs, play a key role in visual cortical plasticity can be traced back more than 25 y to observations that glutamate-stimulated phosphoinositide turnover, mediated in visual cortex by mGluR5 coupled to phospholipase C, is elevated during the postnatal period of heightened sensitivity to MD (19). Early attempts to test this hypothesis were inconclusive owing to the use of weak and nonselective orthosteric compounds (20–22); however, subsequent experiments did confirm that NMDAR-dependent LTD occurs normally in layers 2/3 of visual cortex in Grm5 knockout mice (23).The idea that mGluR5 is critically involved in visual cortical plasticity in vivo was rekindled with the finding that deprived-eye depression fails to occur in layer 4 of Grm5^+/− mutant mice (24). This finding was unexpected because, as reviewed above, a considerable body of evidence has implicated the mechanism of NMDAR-dependent LTD in deprived-eye depression. In the present study, we reexamined the role of mGluR5 in LTD and ocular dominance plasticity in layer 4, using the Grm5^+/− mouse and a highly specific negative allosteric modulator, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), that has proven suitable for chronic inhibition of mGluR5 (25, 26). Our data show that NMDAR-dependent LTD and deprived-eye depression in layer 4 require mGluR5 signaling during postnatal development.     

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.     

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.     

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.     

Long-term depression triggers the selective elimination of weakly integrated synapses

Long-term depression (LTD) weakens synaptic transmission in an activity-dependent manner. It is not clear, however, whether individual synapses are able to maintain a depressed state indefinitely, as intracellular recordings rarely exceed 1 h. Here, we combine optogenetic stimulation of identified Schaffer collateral axons with two-photon imaging of postsynaptic calcium signals and follow the fate of individual synapses for 7 d after LTD induction. Optogenetic stimulation of CA3 pyramidal cells at 1 Hz led to strong and reliable depression of postsynaptic calcium transients in CA1. NMDA receptor activation was necessary for successful induction of LTD. We found that, in the days following LTD, many depressed synapses and their “neighbors” were eliminated from the hippocampal circuit. The average lifetime of synapses on nonstimulated dendritic branches of the same neurons remained unaffected. Persistence of individual depressed synapses was highly correlated with reliability of synaptic transmission, but not with spine size or the amplitude of spine calcium transients. Our data suggest that LTD initially leads to homogeneous depression of synaptic function, followed by selective removal of unreliable synapses and recovery of function in the persistent fraction.Long-lasting modifications of synaptic transmission are thought to underlie learning and information storage in the brain. Intact synaptic plasticity seems to be a precondition for memory formation, and disturbing long-term depression (LTD) or long-term potentiation (LTP) strongly interferes with learning (1–6). Hippocampal field potential recordings have been used to demonstrate that LTD and LTP can be stable for weeks in vivo, at least at the level of large synaptic populations (7–9). It is less clear, however, whether individual synapses can maintain their strength at a specific level over the time scales of memory. Commonly used recording techniques to assess synaptic plasticity (e.g., whole-cell recordings, field recordings, or imaging of Ca²⁺-sensitive dyes) are too short-lived (10–12) or lack single-synapse resolution (7–9). Therefore, it is not known whether the strength of an individual synapse drifts over time, or how specific activity patterns affect the long-term stability of a synapse.In vivo imaging experiments have shown that dendritic spines in mammalian cortex constantly change their morphology and sometimes completely disappear or form de novo (13–15). Life expectancy and turnover of spines is affected by experience and behavioral paradigms, suggesting a regulated, activity-dependent process controlling spine lifetime (16–19). Could LTP and LTD form the missing link between neuronal activity and lasting structural changes? Indeed, induction of LTP at individual excitatory synapses in vitro leads to increased spine head size (12) and insertion of postsynaptic scaffolding proteins (20) and glutamate receptors (21). Potentiation of individual spine synapses selectively increases their stability, pointing to a connection between synaptic plasticity and spine survival (22). Conversely, whether LTD is connected to spine shrinkage or a reduction in spine lifetime is less clear (23–27), as it was not possible to identify individual depressed synapses and to observe them over sufficiently long time periods. Because of these methodological limitations, we know little about LTD at the synaptic level: Are all synapses equally sensitive to depression-inducing activity patterns? For how long do individual synapses stay in the depressed state? Is LTD as synapse-specific as LTP, or does it spread to neighboring synapses?To address these questions, we introduce a noninvasive, optical method based on optogenetic stimulation and two-photon Ca²⁺ imaging to control and measure the activity of individual hippocampal synapses in mature organotypic cultures over a period of 7 d. We found that depressed synapses were frequently eliminated from the circuit in the following days. This delayed elimination was not random: synapses with high release probability were more resistant to elimination than less reliable synapses. Resistant synapses were still connected to the same presynaptic axon after 1 wk and were indistinguishable from nonstimulated synapses, suggesting complete recovery of a subset of initially depressed synapses. Thus, the apparently stable LTD at the level of entire pathways in vivo does not seem to arise from a uniform, long-lasting depression of all stimulated synapses. On the contrary, it might reflect a protracted elimination process that removes a specific subpopulation of all depressed synapses. Our findings suggest that “long-term” depression of Schaffer collateral synapses following optogenetic low-frequency stimulation (oLFS) is, in fact, a transient phenomenon, setting in motion a reorganization of network connectivity.     

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2–related inhibitory peptide; or the blocker of L-voltage–dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.Frey and Morris (1, 2) and their collaborators (3–7) proposed a mechanism whereby relatively “weak” hippocampal long-term potentiation (LTP) or long-term depression (LTD) lasting only a few minutes can nevertheless “tag” the synapses involved with proteins synthesized ad hoc, so that other plasticity-related proteins (PRPs) produced at other sets of synapses by other LTPs or LTDs can be captured by the tagged synapses and strengthen their activity to “long” LTPs or LTDs lasting hours or days (8). LTDs and LTPs can “cross”-tag each other; that is, LTDs can enhance both LTDs and LTPs, and vice versa (6, 8). Because many learned behaviors rely on hippocampal LTP or LTD (7–9), among them the processing of novelty (9, 10) and the making of extinction (11–13), interactions between consecutive learnings can also be explained by the “tagging-and-capture” hypothesis (9, 10, 13), whose application to behavior became known as “behavioral tagging and capture” (5, 7, 9, 13). Typically, exposure to a novel environment [e.g., a nonanxiogenic 50 × 50 × 40-cm open field (OF) (5, 7, 9, 10, 14)] is interpolated before testing for another task, which becomes enhanced (4–10, 13). The usual reaction to novelty is orienting and exploration (14), followed by habituation of this response (14–16). Habituation is perhaps the simplest form of learning, and it consists of inhibition of the orienting/exploratory response (14, 16).We recently showed that the brief exposure of rats to a novel environment (the OF) within a limited time window enhances the extinction of contextual fear conditioning (CFC) through a mechanism of synaptic tagging and capture (13), which is a previously unidentified example of behavioral tagging of inhibitory learning. Fear extinction is most probably due to LTD in the hippocampus (11, 12), although the possibility that it may also involve LTP is not discarded (13). The enhancement of extinction by novelty probably relies on the habituation to the novel environment, which is also probably due to LTD (15, 16). The enhancement of extinction by the exposure to novelty depends on hippocampal gene expression and ribosomal protein synthesis following extinction training and on both ribosomal and nonribosomal protein synthesis caused by the novel experience (13). Nonribosomal protein synthesis that can be blocked by rapamycin is believed to be dendritic (13, 17), so it would be strategically located for tagging-and-capture processes, but it has not been studied in synaptic tagging to date (3–8) or in other forms of behavioral tagging (7–10). As occurs with the interactions between LTPs and/or LTDs (4), the enhancement of extinction by novelty relies on hippocampal but not amygdalar processes (13).Recent findings indicate that several hippocampal processes related to learning and memory, such as the reconsolidation of spatial learning, are highly dependent on NMDA glutamate receptors, calcium/calmodulin protein kinase II (CaMKII), and long-term voltage channel blockers (L-VDCCs), which, in turn, rely on the proteasomal degradation of proteins (18). Here, we study the effects of an NMDA blocker, 2-amino-5-phosphono pentanoic acid (AP5); the L-VDCC blocker nifedipine (Nife); a CaMKII inhibitor, the autocamtide-2–related inhibitory peptide (AIP); and the irreversible proteasome blocker β-lactacystin (12, 13) on the interaction between novelty and extinction (11). As will be seen, we found that both the setting up of tags by extinction and the presumable production of PRPs by the processing of novelty are dependent on NMDA receptors, CaMKII, and L-VDCCs. This endorses and expands the hypothesis that the novelty–extinction interaction relies on synaptic tagging and capture (13).     

From the Cover: Joint CP-AMPA and group I mGlu receptor activation is required for synaptic plasticity in dentate gyrus fast-spiking interneurons

Hippocampal principal cell (PC) assemblies provide the brain with a mnemonic representation of space. It is assumed that the formation of cell assemblies is supported by long-lasting modification of glutamatergic synapses onto perisomatic inhibitory interneurons (PIIs), which provide powerful feedback inhibition to neuronal networks. Repetitive activation of dentate gyrus PIIs by excitatory mossy fiber (MF) inputs induces Hebbian long-term potentiation (LTP). In contrast, long-term depression (LTD) emerges in the absence of PII activity. However, little is known about the molecular mechanisms underlying synaptic plasticity in PIIs. Here, we examined the role of group I metabotropic glutamate receptors 1 and 5 (mGluRs1/5) in inducing plastic changes at MF-PII synapses. We found that mGluRs1/5 are located perisynaptically and that pharmacological block of mGluR1 or mGluR5 abolished MF-LTP. In contrast, their exogenous activation was insufficient to induce MF-LTP but cleared MF-LTD. No LTP could be elicited in PIIs loaded with blockers of G protein signaling and Ca²⁺-dependent PKC. Two-photon imaging revealed that the intracellular Ca²⁺ rise necessary for MF-LTP was largely mediated by Ca²⁺-permeable AMPA receptors (CP-AMPARs), but less by NMDA receptors or mGluRs1/5. Thus, our data indicate that fast Ca²⁺ signaling via CP-AMPARs and slow G protein-mediated signaling via mGluRs1/5 converge to a PKC-dependent molecular pathway to induce Hebbian MF-LTP. We further propose that Hebbian activation of mGluRs1/5 gates PIIs into a “readiness mode” to promote MF-LTP, which, in turn, will support timed PII recruitment, thereby assisting in PC assembly formation.Reorganization of hippocampal principal cell (PC) assemblies during spatial learning is supported by the timed recruitment of GABAergic cells, specifically parvalbumin (PV)-expressing perisomatic inhibitory interneurons (PIIs) (1, 2). Dentate gyrus (DG) PIIs are excited by glutamatergic granule cells (GCs) via mossy fiber (MF) synapses, which can undergo activity-dependent synaptic plasticity (3, 4). Indeed, after long-term potentiation (LTP), a single MF input can reliably activate PIIs (4), indicating that MF-LTP may influence spatial representation in the DG (2, 5).Hebbian MF-PII LTP requires precisely timed pre- and postsynaptic activity (4). It is induced in the PII but is expressed presynaptically (3). MF-LTP requires strong intracellular Ca²⁺ elevation and activation of Ca²⁺-permeable AMPA receptors (CP-AMPARs), but, in contrast to Hebbian LTP at CA1 PII inputs (6), it is independent of NMDA receptors (NMDARs) (4), suggesting that CP-AMPARs could provide the required Ca²⁺ rise for plasticity induction (7, 8). However, recent investigations have proposed a role for group I metabotropic glutamate receptors (mGluRs) in the induction of CP-AMPAR–dependent interneuron plasticity (9–13). Indeed, a Hebbian form of CP-AMPAR–dependent LTP at glutamatergic synapses onto somatostatin (SOM)-expressing CA1 stratum oriens/alveus (O/A) interneurons and at MF inputs onto CA3 interneurons requires mGluR1α activation (9, 11). Furthermore, mGluR1α and mGluR5 contribute to LTP at PC inputs on O/A interneurons, including oriens-lacunosum/moleculare (O-LM) cells (10), but induce long-term depression (LTD) in CA1 fast-spiking GABAergic cells (12). Whether mGluRs1/5 are expressed in hippocampal PV-PIIs remained controversial (14, 15), and their contribution to plasticity in PV-PIIs is unknown. Using whole-cell recordings of GCs paired to PV-PIIs and quantitative immunoelectron microscopy, we show that mGluRs1α/5 are expressed in DG PV-PIIs, contribute to Hebbian LTP, and suppress LTD at their MF inputs. Two-photon (2P) imaging further revealed the main Ca²⁺ sources and Ca²⁺-mediated molecular cascades for MF-LTP induction. Finally, we identify one major molecular mechanism underlying the emergence of MF-LTP.     

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.     

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.     

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).     

Long-term potentiation of glycinergic synapses triggered by interleukin 1β

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABA_A receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.Glycine mediates fast synaptic inhibition throughout the spinal cord, brainstem, and midbrain, controlling normal motor behavior and rhythm generation, somatosensory processing, auditory and retinal signaling, and coordination of reflex responses (1). Strychnine-sensitive glycine receptors (GlyRs) are pentameric ligand-gated chloride channels of the Cys-loop receptor family that together with GABA_A receptors (GABA_ARs) dynamically interact with the synaptic scaffold protein gephyrin to form inhibitory synapses (1, 2). In the dorsal horn of the spinal cord, glycinergic synapses are essential for nociceptive and tactile sensory processing both during adaptive and pathological pain states (3–7). However, compared with glutamatergic and GABAergic synapses, little is known about the regulation of their synaptic strength. Several studies have examined glycine receptor trafficking in cultured neurons and in heterologous expression systems (8, 9). Intracellular Ca²⁺ appears important in the stabilization of GlyRs at synapses in culture (10), and elevation of intracellular Ca²⁺ can also potently increase glycine receptor single channel openings in cultured cells and in heterologous systems (11). However, the modulation of glycinergic synaptic strength in native tissue remains relatively unexplored.Following peripheral injury or inflammation, changes in tactile perception develop, including hyperalgesia (exaggerated pain upon noxious stimulation), allodynia (pain in response to innocuous stimuli), and secondary hyperalgesia (pain spreading beyond the confines of the injured region). Inhibitory interneurons of the spinal dorsal horn have been proposed to gate the flow of innocuous and nociceptive sensory information from the periphery to higher brain centers (12), and supportive evidence for this idea is growing (13–17). Loss of GABAergic/glycinergic inhibition contributes to enhanced transmission of nociceptive signals through the dorsal horn circuit during pain states, resulting in hyperalgesia and allodynia (3, 18–20). For example, polysynaptic A-fiber inputs onto neurokinin 1 receptor (NK1R)-expressing projection neurons become apparent only when GABA_AR and GlyRs are pharmacologically blocked, indicating that under conditions of disinhibition, nonnoxious mechanical stimuli can drive nociceptive-specific projection pathways and elicit allodynia (21). The majority of neurons tested in the dorsal horn receive glycinergic synapses, including lamina I projection neurons, both excitatory and inhibitory interneurons of lamina II (22, 23), and inhibitory glycinergic neurons (24). Given the diversity of afferent targets, it is likely that glycinergic synapses are differentially modulated in a cell type- and subregion-specific manner. For example, during chronic inflammation, prostaglandin E₂ selectively depresses glycinergic synaptic inputs onto nonglycinergic neurons (24). Similarly, peripheral nerve injury suppresses glycinergic inhibition of a specific excitatory interneuron class [protein kinase C (PKC)γ⁺ neurons receiving Aβ fiber inputs], allowing excitatory afferents carrying nonnociceptive tactile information to activate ascending projections of nociceptive pathways that are normally under strong inhibitory control (23).Both hyperalgesia and allodynia occur within minutes of peripheral inflammation, but the mechanisms underlying these rapid perceptual alterations are poorly understood. The proinflammatory cytokine, IL-1β, is a potent hyperalgesic agent (25–27), contributing both to peripheral and central sensitization after tissue damage (28–31). Following tissue trauma, nerve injury, or inflammation, IL-1β levels are up-regulated in the spinal cord itself (29, 32, 33), and delivery of IL-1β intrathecally increases the activity of superficial dorsal horn neurons that transmit pain signals to the brain (34, 35). Intrathecal delivery of an IL-1 receptor antagonist blocks allodynia in rodent models of inflammatory pain (36, 37). A recent study also found that IL-1β application rapidly potentiated primary afferent (glutamatergic) synapses in dorsal horn slices, through unidentified signaling molecules released from glial cells (38). Here we report that IL-1β rapidly elicits a postsynaptic form of long-term potentiation (LTP) at glycinergic synapses on lamina II inhibitory neurons (GlyR LTP), and that the same glycinergic synapses are potentiated after peripheral inflammation.     

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice

Mutations that lead to Huntington’s disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M₄ subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M₄ positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M₄ PAMs could provide a therapeutic strategy for the treatment of HD.Huntington’s disease (HD) is a rare and fatal neurodegenerative disease caused by an expansion of a CAG triplet repeat in Htt, the gene that encodes for the protein huntingtin (1, 2). HD is characterized by a prediagnostic phase that includes subtle changes in personality, cognition, and motor function, followed by a more severe symptomatic stage initially characterized by hyperkinesia (chorea), motor incoordination, deterioration of cognitive abilities, and psychiatric symptoms. At later stages of disease progression, patients experience dystonia, rigidity, and bradykinesia, and ultimately death (3–7). The cortex and striatum are the most severely affected brain regions in HD and, interestingly, an increasing number of reports suggest that alterations in cortical and striatal physiology are present in prediagnostic individuals and in young HD mice (6–16).Striatal spiny projection neurons (SPNs) receive large glutamatergic inputs from the cortex and thalamus, as well as dopaminergic innervation from the substantia nigra. In the healthy striatum, the interplay of these neurotransmitters coordinates the activity of SPNs and striatal interneurons, regulating motor planning and execution as well as cognition and motivation (17, 18). Htt mutations lead to an early increase in striatal glutamatergic transmission, which begins during the asymptomatic phase of HD (12–14) and could contribute to synaptic changes observed in later stages of HD (19, 20). Based on this, pharmacological agents that reduce excitatory transmission in the striatum could reduce or prevent the progression of alterations in striatal synaptic function and behavior observed in symptomatic stages of HD.Muscarinic acetylcholine receptors (mAChRs), particularly M₄, can inhibit transmission at corticostriatal synapses (21–25). Therefore, it is possible that selective activation of specific mAChR subtypes could normalize excessive corticostriatal transmission in HD. Interestingly, previous studies also suggest that HD is associated with alterations of striatal cholinergic markers, including mAChRs (26–29). We now provide exciting new evidence that M₄-mediated control of corticostriatal transmission is increased in young asymptomatic HD mice and that M₄ positive allosteric modulators (PAMs) may represent a new treatment strategy for normalizing early changes in corticostriatal transmission and reducing the progression of HD.     

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.The human airway is normally protected from injury caused by microbial colonization and viral infection by a complex immune defense system. The cornerstone of airway defense is mucociliary clearance. Particles, including bacteria, are captured in mucus and removed by an efficient mucociliary clearance mechanism. Airway host defense is compromised in individuals with cystic fibrosis (CF), whose lungs are thus prone to chronic bacterial infections, frequently with Pseudomonas aeruginosa, and inflammation that may eventually cause lung tissue damage and respiratory failure (1, 2). The events leading from cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation to airway disease are incompletely understood, but accumulating evidence suggests that CF airway disease results from abnormal microbial clearance (3, 4).Although chronic inflammation is a major aspect of CF lung disease, recent data show that the initial consequence of CFTR mutation is impaired ability to eradicate bacteria. In previous studies, lungs from animal models of CF (F508del and CFTR^−/− pigs) (5, 6) did not eradicate bacteria as effectively as lungs from WT littermates before the development of inflammation (3, 4). These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs (4).The failure to clear bacteria likely results from abnormal airway surface liquid (ASL) secretion and properties (6–10). The ASL consists of a layer of mucus that traps inhaled particles and a periciliary liquid layer that keeps the mucus an optimum distance from the underlying epithelia to maximize ciliary mobility (10, 11). The mucus layer is a complex mixture of water, salts, gel-forming mucins, and antimicrobial compounds that helps inactivate, kill, and trap pathogens and facilitates mucociliary clearance (10, 11). In CF airways, both the bacteria-killing properties and ASL secretion are abnormal (3, 9). The airway liquid produced by CFTR^−/− swine has weaker bactericidal properties compared with that produced by WT littermates, owing to abnormal pH (3, 4). In addition, human CF airways, 1-d-old CF piglets, newborn CFTR^−/− ferrets, and CFTR^−/− mice fail to respond to stimulatory signals that normally elicit strong ASL secretion (6–9). Consequently, it has been proposed that abnormal secretion of fluid and mucin in response to bacterial infection may contribute to the pathogenesis of CF lung disease (7–10, 12–15); however, the central questions of whether bacteria trigger ASL secretion in the airways, and the role of CFTR in such a process, have not been explored previously, owing to the lack of a suitable experimental technique.We have developed a novel synchrotron-based method to measure the height of the ASL layer covering the epithelium of intact, isolated swine trachea. We show that the introduction of P. aeruginosa into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This is a local response that affects only the glands in close proximity to the bacteria and requires expression of the bacterial protein flagellin. We also show that Staphylococcus aureus and Haemophilus influenzae trigger CFTR-dependent ASL secretion, indicating that this response is not unique to P. aeruginosa. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion by submucosal glands, facilitating infection and inflammation.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Thalamic model of awake alpha oscillations and implications for stimulus processing

We describe a unique conductance-based model of awake thalamic alpha and some of its implications for function. The full model includes a model for a specialized class of high-threshold thalamocortical cells (HTC cells), which burst at the alpha frequency at depolarized membrane potentials (∼−56 mV). Our model generates alpha activity when the actions of either muscarinic acetylcholine receptor (mAChR) or metabotropic glutamate receptor 1 (mGluR1) agonists on thalamic reticular (RE), thalamocortical (TC), and HTC cells are mimicked. In our model of mGluR1-induced alpha, TC cells are equally likely to fire during any phase of alpha, consistent with in vitro experiments. By contrast, in our model of mAChR-induced alpha, TC cells tend to fire either at the peak or the trough of alpha, depending on conditions. Our modeling suggests that low levels of mGluR1 activation on a background of mAChR agonists may be able to initiate alpha activity that biases TC cells to fire at certain phases of alpha, offering a pathway for cortical control. If we introduce a strong stimulus by increasing the frequency of excitatory postsynaptic potentials (EPSPs) to TC cells, an increase in alpha power is needed to mimic the level of phasing of TC cells observed in vivo. This increased alpha power reduces the probability that TC cells spike near the trough of alpha. We suggest that mAChR-induced alpha may contribute to grouping TC activity into discrete perceptual units for processing, whereas mGluR1-induced alpha may serve the purpose of blocking unwanted stimuli from reaching the cortex.Alpha rhythms (8–13 Hz) were first observed in humans over the occipital cortex by Berger (1), when subjects were in a relaxed state with their eyes closed. Occipital alpha has been investigated extensively since. However, alpha rhythms are not strictly confined to this area of cortex; alpha activity has also been reported in the somatosensory cortex (2), the auditory cortex (3), and the prefrontal cortex (4).Both the neural substrates responsible for the genesis of alpha and its functional role in cognition remain hotly debated. At the functional level, the point of contention is whether alpha activity serves to process information relevant to the task at hand or serves to filter out irrelevant information. The debate over where alpha activity is generated primarily revolves around whether it is generated by the neocortex, by the thalamus, or by a combination of the two. We make use of recent findings (5–10), as well as prior findings (11–17), to construct a unique conductance-based thalamic model of awake alpha, and use it to address the above controversy.Studies have found that during simultaneous in vivo recordings from the thalamus and neocortex, alpha activity in the neocortex is accompanied by alpha rhythms in the local field potential of the thalamus and in the firing patterns of individual thalamocortical (TC) cells (5, 18). During alpha activity, only 10–30% of TC cells fire in the alpha frequency (5, 6). Their firing pattern consists of high-threshold bursts (HTBs), with the intervals between bursts occurring at the alpha frequency, and gap junctions play a critical role in synchronizing their activity (10). Alpha activity can be induced in thalamic slices in the presence of metabotropic glutamate receptor (mGluR1) agonists (5) or muscarinic acetylcholine receptor (mAChR) agonists (8). As in the in vivo case, only a small fraction of TC cells exhibits HTB at the alpha frequency in the presence of mGluR1 and mAChR agonists. Although the mechanisms by which mGluR1 agonists and mAChR agonists induce HTB may differ, they both seem to do so, in part, by reducing potassium leak conductances and by activating an I_T channel that acts at more depolarized membrane potentials than the standard I_T channel (7).For our model, we developed two submodels: one for a specialized class of high-threshold thalamocortical cells (HTC cells) and one for an I_T-channel variant suggested to play a critical role in the generation of thalamic alpha (5–7, 17). We denote by I_TLT and I_THT the calcium currents associated with the low- and high-threshold variants, respectively. The model generates alpha activity upon choosing parameters to reflect the presence of either mGluR1 or mAChR, consistent with experimental data (5–9). We show that mGluR1- and mAChR-mediated alpha rhythms produce differential effects on the firing of TC cells with respect to the alpha rhythm, and discuss the functional implications.     

Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

Dystroglycan mediates homeostatic synaptic plasticity at GABAergic synapses

Dystroglycan (DG), a cell adhesion molecule well known to be essential for skeletal muscle integrity and formation of neuromuscular synapses, is also present at inhibitory synapses in the central nervous system. Mutations that affect DG function not only result in muscular dystrophies, but also in severe cognitive deficits and epilepsy. Here we demonstrate a role of DG during activity-dependent homeostatic regulation of hippocampal inhibitory synapses. Prolonged elevation of neuronal activity up-regulates DG expression and glycosylation, and its localization to inhibitory synapses. Inhibition of protein synthesis prevents the activity-dependent increase in synaptic DG and GABA_A receptors (GABA_ARs), as well as the homeostatic scaling up of GABAergic synaptic transmission. RNAi-mediated knockdown of DG blocks homeostatic scaling up of inhibitory synaptic strength, as does knockdown of like-acetylglucosaminyltransferase (LARGE)—a glycosyltransferase critical for DG function. In contrast, DG is not required for the bicuculline-induced scaling down of excitatory synaptic strength or the tetrodotoxin-induced scaling down of inhibitory synaptic strength. The DG ligand agrin increases GABAergic synaptic strength in a DG-dependent manner that mimics homeostatic scaling up induced by increased activity, indicating that activation of this pathway alone is sufficient to regulate GABA_AR trafficking. These data demonstrate that DG is regulated in a physiologically relevant manner in neurons and that DG and its glycosylation are essential for homeostatic plasticity at inhibitory synapses.Muscular dystrophies are often associated with mild to severe cognitive deficits, epilepsy, and other neurological deficits (1–3). This is particularly evident in muscular dystrophies caused by mutations that affect glycosylation of the membrane glycoprotein α-dystroglycan (α-DG) (4). α-DG docks with transmembrane β-DG to form the functional core of the dystrophin-associated glycoprotein complex (DGC) that links adhesive proteins in the extracellular matrix to dystrophin (5). α-DG is heavily glycosylated and interacts via its carbohydrate side chains with laminin and laminin G-like domains in a variety of proteins including agrin, perlecan, slit, neurexin, and pikachurin (6–10). Key carbohydrate residues are added onto α-DG by several glycosyltransferases, most notably like-acetylglucosaminyltransferase (LARGE) (11). LARGE is necessary for functional glycosylation of α-DG (12), and is mutated in muscular dystrophies associated with severe cognitive deficits (4).DG was first identified in the nervous system (13), where it is important during development for neuroblast migration (14), axon guidance (7), and ribbon synapse formation (8). At neuromuscular synapses, DG is required for the stabilization of acetylcholine receptors in the postsynaptic density and contributes to the accumulation of acetylcholinesterase (10, 15). However, the function of DG at central synapses remains essentially unknown. In the mature central nervous system (CNS), neuronal DGC components are exclusively colocalized with GABA_A receptors (GABA_ARs) in multiple brain regions (16–18), raising the possibility for a role in GABA_AR regulation. However, DG is dispensable for GABAergic synapse formation in hippocampal cultures (17), although adult mice lacking full-length dystrophin show reduced clustering of GABA_ARs in the hippocampus and other brain regions (16, 19, 20). Because dystrophin localization at GABAergic synapses depends on DG (17), these findings suggest that DG may regulate the plasticity of mature GABAergic synapses. Homeostatic synaptic plasticity is widely thought to be essential for brain function and involves the reciprocal regulation of glutamatergic and GABAergic synapses to stabilize neuronal activity (21). Chronic elevation of neuronal activity is associated with an increase in synaptic GABA_ARs (22, 23), but the mechanistic details are incompletely understood.Here, we assess the roles of DG and α-DG glycosylation in regulating the expression of homeostatic synaptic plasticity at GABAergic synapses. We find that in mature hippocampal cultures, prolonged elevation of neuronal activity up-regulates DG expression and the coclustering of α-DG and GABA_ARs. Inhibition of protein synthesis or knockdown of DG blocks homeostatic scaling up of GABAergic synaptic strength. Knockdown of the selective α-DG glycosyltransferase LARGE also blocks homeostatic scaling up, suggesting a role for ligand binding. Furthermore, exogenous application of agrin—a ligand for glycosylated α-DG—is sufficient to scale up GABAergic synaptic strength in a DG-dependent fashion. These data identify a mechanism whereby expression of glycosylated α-DG is linked to neuronal activity level and is essential for homeostatic scaling up of GABAergic synaptic strength by regulating GABA_AR abundance at the synapse.     

AMPA receptor inhibition by synaptically released zinc

The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses.The development, function, and experience-dependent plasticity of the mammalian brain depend on the refined neuronal interactions that occur in synapses. In the majority of excitatory synapses, the release of the neurotransmitter glutamate from presynaptic neurons opens transmembrane ion channels in postsynaptic neurons, the ionotropic glutamate receptors, thereby generating the flow of excitatory signaling in the brain. As a result, these receptors play a fundamental role in normal function and development of the brain, and they are also involved in many brain disorders (1).The ionotropic glutamate receptor family consists of AMPA, kainate, and NMDA receptors (NMDARs). Although kainate receptor-mediated excitatory postsynaptic responses occur in a few central synapses (2), AMPA receptors (AMPARs) and NMDARs are localized in the postsynaptic density of the vast majority of glutamatergic synapses in the brain, mediating most of excitatory neurotransmission (1). NMDAR function is regulated by a wide spectrum of endogenous allosteric neuromodulators that fine-tune synaptic responses (3–5); however, much less is known about endogenous AMPAR neuromodulators [(1, 5), but see refs. 6 and 7]. Recent structural studies revealed that the amino terminal domain (ATD) and ligand-binding domain (LBD) are tightly packed in NMDARs but not AMPARs (8–10). These structural differences explain some of the functional differences in allosteric modulation between AMPARs and NMDARs, such as why the ATD of NMDARs, unlike that of AMPARs, modulates function and contains numerous binding sites for allosteric regulators. Nonetheless, given the importance of fine-tuning both synaptic AMPAR and NMDAR responses for brain function, it is puzzling that there is not much evidence for endogenous, extracellular AMPAR modulation. The discovery and establishment of endogenous AMPAR modulators is crucial both for understanding ionotropic glutamate receptor signaling and for developing therapeutic agents for the treatment of AMPAR-related disorders, such as depression, cognitive dysfunctions associated with Alzheimer’s disease, and schizophrenia (1, 11).Free, or readily chelatable, zinc is an endogenous modulator of synaptic and extrasynaptic NMDARs (12–15). Free zinc is stored in glutamatergic vesicles in many excitatory synapses in the cerebral cortex, limbic, and brainstem nuclei (16). In some brain areas, such as in the hippocampus, 50% of boutons synapsing onto CA1 neurons and all mossy fibers synapsing onto CA3 neurons contain synaptic free zinc (17). Whereas earlier studies demonstrated that exogenous zinc inhibits AMPARs (18–21), more recent work suggests that endogenously released synaptic zinc does not modulate AMPARs in central synapses (14, 22). This conclusion was derived from the inability to efficiently chelate and quantify synaptic zinc with the zinc-selective chelators and probes used (15), in apparent support of the hypothesized low levels of released zinc during synaptic stimulation (14).Recent work in our laboratories used new chemical tools that allowed us to intercept and visualize mobile zinc efficiently (15). These studies revealed modulation of extrasynaptic NMDARs by zinc and led us to reinvestigate whether synaptically released zinc might be an endogenous modulator of AMPARs as well. In the present study, we applied these same tools in electrophysiological, laser-based glutamate uncaging and in imaging experiments using wild type and genetically modified mice that lack synaptic zinc.