Synapse composition and organization following chronic activity blockade in cultured hippocampal neurons

Activity plays multiple roles in the expression of synaptic plasticity, and has been shown to regulate the localization of both neurotransmitter receptors and downstream signaling machinery. However, the role of activity in central synapse formation and organization is incompletely understood. Some studies indicate that synapse formation can occur in the absence of synaptic activity, while others indicate that activity is required for synapse maintenance and receptor recruitment. In addition, the effects of long-term blockade of transmission generally, rather than blockade of specific receptors, on postsynaptic protein complement has been poorly characterized. In order to address the role of activity in synapse formation and postsynaptic specialization, we used tetanus toxin to chronically cleave VAMP2 and inhibit SNARE-mediated neurotransmitter release in cultured hippocampal neurons. Although these neurons are deficient in synaptic release, they are of normal size and morphology. In addition, both excitatory and inhibitory synapses form along their processes with normal density. These synapses have a remarkably similar cellular and molecular organization compared to controls, and are capable of recruiting postsynaptic scaffolding proteins, GABA, and glutamate receptors. Subcellular enrichment of synaptic proteins into specialized domains also appears intact. These data indicate that global activity inhibition is insufficient to disrupt central synapse formation or organization.     

The gray area between synapse structure and function-Gray's synapse types I and II revisited

On the basis of ultrastructural parameters, the concept was formulated that asymmetric Type I and symmetric Type II synapses are excitatory and inhibitory, respectively. This "functional Gray synapses concept" received strong support from the demonstration of the excitatory neurotransmitter glutamate in Type I synapses and of the inhibitory neurotransmitter γ-aminobutyric acid in Type II synapses, and is still frequently used in modern literature. However, morphological and functional evidence has accumulated that the concept is less tenable. Typical features of synapses like shape and size of presynaptic vesicles and synaptic cleft and presence of a postsynaptic density (PsD) do not always fit the postulated (excitatory/inhibitory) function of Gray's synapses. Furthermore, synapse function depends on postsynaptic receptors and associated signal transduction mechanisms rather than on presynaptic morphology and neurotransmitter type. Moreover, the notion that many synapses are difficult to classify as either asymmetric or symmetric has cast doubt on the assumption that the presence of a PsD is a sign of excitatory synaptic transmission. In view of the morphological similarities of the PsD in asymmetric synapses with membrane junctional structures such as the zonula adherens and the desmosome, asymmetric synapses may play a role as links between the postsynaptic and presynaptic membrane, thus ensuring long-term maintenance of interneuronal communication. Symmetric synapses, on the other hand, might be sites of transient communication as takes place during development, learning, memory formation, and pathogenesis of brain disorders. Confirmation of this idea might help to return the functional Gray synapse concept its central place in neuroscience.     

Postsynaptic gephyrin clustering controls the development of adult‐born granule cells in the olfactory bulb

In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin—the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density—and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult‐born GCs, we observed that overexpression of the phospho‐deficient gephyrin mutant eGFP‐gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of α2‐GABA_ARs. In contrast, overexpression of the dominant‐negative eGFP‐gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long‐term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP‐gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long‐term survival of adult‐born olfactory bulb neurons. J. Comp. Neurol. 523:1998–2016, 2015 © 2015 Wiley Periodicals, Inc.     

Surface trafficking of receptors between synaptic and extrasynaptic membranes: and yet they do move!

Concentration of neurotransmitter receptors at synapses is thought to result from stable binding to subsynaptic scaffold proteins. Recent data on synaptic plasticity have shown that changes in synaptic strength derive partly from modification of postsynaptic receptor numbers. This has led to the notion of receptor trafficking into and out of synapses. The proposed underlying mechanisms have under-evaluated the role of extrasynaptic receptors. Recent technological advances have allowed imaging of receptor movements at the single-molecule level, and these experiments demonstrate that receptors switch at unexpected rates between extrasynaptic and synaptic localizations by lateral diffusion. Variation in receptor numbers at postsynaptic sites is therefore likely to depend on regulation of diffusion by modification of the structure of the membrane and/or by transient interactions with scaffolding proteins. This review is part of the TINS Synaptic Connectivity series.     

A specific GABAergic synapse onto oligodendrocyte precursors does not regulate cortical oligodendrogenesis

In the brain, neurons establish bona fide synapses onto oligodendrocyte precursor cells (OPCs), but the function of these neuron‐glia synapses remains unresolved. A leading hypothesis suggests that these synapses regulate OPC proliferation and differentiation. However, a causal link between synaptic activity and OPC cellular dynamics is still missing. In the developing somatosensory cortex, OPCs receive a major type of synapse from GABAergic interneurons that is mediated by postsynaptic γ2‐containing GABA_A receptors. Here we genetically silenced these receptors in OPCs during the critical period of cortical oligodendrogenesis. We found that the inactivation of γ2‐mediated synapses does not impact OPC proliferation and differentiation or the propensity of OPCs to myelinate their presynaptic interneurons. However, this inactivation causes a progressive and specific depletion of the OPC pool that lacks γ2‐mediated synaptic activity without affecting the oligodendrocyte production. Our results show that, during cortical development, the γ2‐mediated interneuron‐to‐OPC synapses do not play a role in oligodendrogenesis and suggest that these synapses finely tune OPC self‐maintenance capacity. They also open the interesting possibility that a particular synaptic signaling onto OPCs plays a specific role in OPC function according to the neurotransmitter released, the identity of presynaptic neurons or the postsynaptic receptors involved.     

Neuronal sandwiches: a method for rapid and controlled initiation of synapses

Synapse formation is a fast, dynamic process that involves the assembly of many molecules following axodendritic contact. Neuronal cultures are often used to study the insertion of fluorescently tagged pre- and postsynaptic molecules in vitro. However, this task still remains challenging, since the time-point and location of newly forming synapses are largely unpredictable and rely on random contact events. We developed a technique that controls the time-point of interaction between axons and dendrites, and thus the onset of synapse formation. Dissociated hippocampal neurons were cultivated on two different coverslips, allowing for the separate outgrowth of axonal networks and of neurons with sparsely innervated dendrites. Pre- and postsynaptic partners were brought in contact as coverslips were merged. Time-lapse imaging showed clustering of GFP/PSD-95 in postsynaptic neurons within 1-3h, indicating the rapid formation of new synaptic sites. Localization of DsRed, as a control protein, remained unchanged. Imaging of neuronal activity using calcium sensitive dyes revealed that in a number of cases neurons of the pre- and postsynaptic layer were synchronously active, suggesting the functionality of newly formed synapses across layers. Therefore, our new method is a valuable tool to control synapse formation and for investigating the temporal role of signaling molecules during this process.     

Ephrin regulation of synapse formation, function and plasticity

Synapses enable the transmission of information within neural circuits and allow the brain to change in response to experience. During the last decade numerous proteins that can induce synapse formation have been identified. Many of these synaptic inducers rely on trans-synaptic cell-cell interactions to generate functional contacts. Moreover, evidence now suggests that the same proteins that function early in development to regulate synapse formation may help to maintain and/or regulate the function and plasticity of mature synapses. One set of receptors and ligands that appear to impact both the development and the mature function of synapses are Eph receptors (erythropoietin-producing human hepatocellular carcinoma cell line) and their surface associated ligands, ephrins (Eph family receptor interacting proteins). Ephs can initiate new synaptic contacts, recruit and stabilize glutamate receptors at nascent synapses and regulate dendritic spine morphology. Recent evidence demonstrates that ephrin ligands also play major roles at synapses. Activation of ephrins by Eph receptors can induce synapse formation and spine morphogenesis, whereas in the mature nervous system ephrin signaling modulates synaptic function and long-term changes in synaptic strength. In this review we will summarize the recent progress in understanding the role of ephrins in presynaptic and postsynaptic differentiation, and synapse development, function and plasticity.     

Differential regulation of GABA(A) receptor and gephyrin postsynaptic clustering in immature hippocampal neuronal cultures

Gephyrin is a postsynaptic scaffolding protein involved in clustering of glycine- and GABA(A) receptors at inhibitory synapses. The role of gephyrin in GABAergic synapses, the nature of its interactions with GABA(A) receptors, and the mechanisms of targeting to GABAergic synapses are largely unknown. To gain further insights into these questions, the formation of GABA(A) receptor and gephyrin clusters and their distribution relative to presynaptic terminals were investigated in immature cultures of embryonic hippocampal neurons using triple immunofluorescence staining. GABA(A) receptor clusters, labeled for the alpha2 subunit, formed independently of gephyrin clusters, and were distributed on neurites at constant densities, either extrasynaptically or, to a lesser extent, postsynaptically, apposed to synapsin-I-positive axon terminals. In contrast, gephyrin clusters were always associated with GABA(A) receptors and were preferentially localized postsynaptically. Their density increased linearly with the extent of innervation, which developed rapidly during the first week in vitro. These results suggested that GABA(A) receptor clustering is mediated by cell-autonomous mechanisms independent of synapse formation. Their association with gephyrin is dynamically regulated and may contribute to stabilization at postsynaptic sites. Labeling for vesicular glutamate transporters revealed that most synapses in these immature cultures were presumably glutamatergic, implying that postsynaptic GABA(A) receptor and gephyrin clusters initially were located in "mismatched" synapses. However, clusters appropriately localized in GABAergic synapses were distinctly larger and more intensely stained. Altogether, these results demonstrate that the targeting of GABA(A) receptor and gephyrin clusters to GABAergic synapses occurs secondarily and is regulated by presynaptic factors that are not essential for clustering.     

Synaptogenesis in the cerebellar cortex: differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABA(A) receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABA(A) receptors and gephyrin co-aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABA(A) receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABA(A) receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells.     

Neuregulin1 downregulates postsynaptic GABAA receptors at the hippocampal inhibitory synapse

The growth factor neuregulin 1 (NRG1) has been proposed to contribute to the formation and maturation of neuromuscular and interneuronal synapses by upregulating the expression of specific neurotransmitter receptor subunits. In the present report, we show that, in the hippocampus, NRG1 is expressed in a pattern suggesting that it regulates synapse development in the CA1 region. However, in contrast to what has been shown in other synapses, NRG1 reduces the expression of gamma-aminobutyric acid (GABA)A receptors alpha subunits in hippocampal slices, and the mean amplitude of GABAergic miniature inhibitory postsynaptic currents (IPSCs) in hippocampal CA1 pyramidal neurons, without affecting IPSC kinetics or frequency. These effects of NRG1 occur without concomitant changes in glutamate receptors and other synaptic proteins. We propose that the role of NRG1 in the formation and maturation in the hippocampal inhibitory synapse is downregulation, rather than upregulation, of receptor subunit expression. These results suggest that NRG1 may contribute to the reduction in GABAergic synaptic activity in hippocampal CA1 pyramidal neurons that normally occurs during early postnatal development, and that alterations in NRG1 signaling in the hippocampus may contribute to schizophrenia and epilepsy.     

Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function

Cell adhesion represents the most direct way of coordinating synaptic connectivity in the brain. Recent evidence highlights the importance of a trans-synaptic interaction between postsynaptic neuroligins and presynaptic neurexins. These transmembrane molecules bind each other extracellularly to promote adhesion between dendrites and axons. This signals the recruitment of presynaptic and postsynaptic molecules to form a functional synapse. Remarkably, neuroligins alone can induce the formation of fully functional presynaptic terminals in contacting axons. Conversely, neurexins alone can induce postsynaptic differentiation and clustering of receptors in dendrites. Therefore, the neuroligin-neurexin interaction has the unique ability to act as a bi-directional trigger of synapse formation. Here, we review several recent studies that offer clues as to how these proteins form synapses and how they might function in the brain to establish and modify neuronal network properties and cognition.     

Endocannabinoids and synaptic function in the CNS.

Marijuana affects neural functions through the binding of its active component (Delta(9)-THC) to cannabinoid receptors in the CNS. Recent studies have elucidated that endogenous ligands for cannabinoid receptors, endocannabinoids, serve as retrograde messengers at central synapses. Endocannabinoids are produced on demand in activity-dependent manners and released from postsynaptic neurons. The released endocannabinoids travel backward across the synapse, activate presynaptic CB1 cannabinoid receptors, and modulate presynaptic functions. Retrograde endocannabinoid signaling is crucial for certain forms of short-term and long-term synaptic plasticity at excitatory or inhibitory synapses in many brain regions, and thereby contributes to various aspects of brain function including learning and memory. Molecular identities of the CB1 receptor and enzymes involved in production and degradation of endocannabinoids have been elucidated. Anatomical studies have demonstrated unique distributions of these molecules around synapses, which provide morphological bases for the roles of endocannabinoids as retrograde messengers. CB1-knockout mice exhibit various behavioral abnormalities and multiple defects in synaptic plasticity, supporting the notion that endocannabinoid signaling is involved in various aspects of neural function. In this review article, the authors describe molecular mechanisms of the endocannabinoid-mediated synaptic modulation and its possible physiological significance.     

Quantification of postsynaptic density proteins: glutamate receptor subunits and scaffolding proteins

The postsynaptic density (PSD) protein complex has long been a major target of proteomics in neuroscience. As the number of glutamate receptors on a synapse is one of the main determinants of synaptic efficacy, determining the absolute numbers of receptors in the PSD is necessary for estimating the amplitude of the excitatory postsynaptic current (EPSC) in individual synapses. Moreover, as the receptor molecules are embedded in a macromolecular complex within the PSD, stoichiometry between the receptors and other PSD proteins could help explain the functional and regional specialization of the synapses and their possible roles in synaptic plasticity. Here, I review various studies concerned with the quantification of PSD proteins.     

Biglycan is an extracellular MuSK binding protein important for synapse stability

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.     

Overexpression of the cell adhesion protein neuroligin‐1 induces learning deficits and impairs synaptic plasticity by altering the ratio of excitation to inhibition in the hippocampus

Trans‐synaptic cell‐adhesion molecules have been implicated in regulating CNS synaptogenesis. Among these, the Neuroligin (NL) family (NLs 1–4) of postsynaptic adhesion proteins has been shown to promote the development and specification of excitatory versus inhibitory synapses. NLs form a heterophilic complex with the presynaptic transmembrane protein Neurexin (NRX). A differential association of NLs with postsynaptic scaffolding proteins and NRX isoforms has been suggested to regulate the ratio of excitatory to inhibitory synapses (E/I ratio). Using transgenic mice, we have tested this hypothesis by overexpressing NL1 in vivo to determine whether the relative levels of these cell adhesion molecules may influence synapse maturation, long‐term potentiation (LTP), and/or learning. We found that NL1‐overexpressing mice show significant deficits in memory acquisition, but not in memory retrieval. Golgi and electron microscopy analysis revealed changes in synapse morphology indicative of increased maturation of excitatory synapses. In parallel, electrophysiological examination indicated a shift in the synaptic activity toward increased excitation as well as impairment in LTP induction. Our results demonstrate that altered balance in the expression of molecules necessary for synapse specification and development (such as NL1) can lead to defects in memory formation and synaptic plasticity and outline the importance of rigidly controlled synaptic maturation processes. © 2009 Wiley‐Liss, Inc.     

Gephyrin clustering is required for the stability of GABAergic synapses

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transsynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.     

CASK and Dlg form a PDZ protein complex at the mammalian neuromuscular junction

Membrane-associated guanylate kinases (MAGUKs) are modular adapter proteins that serve as scaffolding molecules and anchor channels and receptors via their PDZ (PSD-95, Dlg, Zo-1) domains. Calcium, calmodulin-associated serine/threonine kinase (CASK) is a MAGUK that is critical at synapses in the central nervous system and at cell-cell junctions because of its interactions with channels, receptors, and structural proteins. We show via confocal microscopy that CASK and another MAGUK, Discs Large (Dlg), are present at the mammalian neuromuscular junction in skeletal muscle. Immunoprecipitation data from mouse muscle show that CASK associates with Dlg, providing evidence of a MAGUK protein complex at this synapse. These data indicate that CASK and Dlg may act as a scaffold for organizing receptors and channels at the postsynaptic membrane of the neuromuscular junction.     

Afadin, a Ras/Rap effector that controls cadherin function, promotes spine and excitatory synapse density in the hippocampus

Many molecules regulate synaptogenesis, but intracellular signaling pathways required for their functions are poorly understood. Afadin is a Rap-regulated, actin-binding protein that promotes cadherin complex assembly as well as binding many other cell adhesion molecules and receptors. To examine its role in mediating synaptogenesis, we deleted afadin (mllt1), using a conditional allele, in postmitotic hippocampal neurons. Consistent with its role in promoting cadherin recruitment, afadin deletion resulted in 70% fewer and less intense N-cadherin puncta with similar reductions of β-catenin and αN-catenin puncta densities and 35% reduction in EphB2 puncta density. Its absence also resulted in 40% decreases in spine and excitatory synapse densities in the stratum radiatum of CA1, as determined by morphology, apposition of presynaptic and postsynaptic markers, and synaptic transmission. The remaining synapses appeared to function normally. Thus, afadin is a key intracellular signaling molecule for cadherin recruitment and is necessary for spine and synapse formation in vivo.     

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.     

N‐cadherin regulates molecular organization of excitatory and inhibitory synaptic circuits in adult hippocampus in vivo

N‐Cadherin and β‐catenin form a transsynaptic adhesion complex required for spine and synapse development. In adulthood, N‐cadherin mediates persistent synaptic plasticity, but whether the role of N‐cadherin at mature synapses is similar to that at developing synapses is unclear. To address this, we conditionally ablated N‐cadherin from excitatory forebrain synapses in mice starting in late postnatal life and examined hippocampal structure and function in adulthood. In the absence of N‐cadherin, β‐catenin levels were reduced, but numbers of excitatory synapses were unchanged, and there was no impact on number or shape of dendrites or spines. However, the composition of synaptic molecules was altered. Levels of GluA1 and its scaffolding protein PSD95 were diminished and the density of immunolabeled puncta was decreased, without effects on other glutamate receptors and their scaffolding proteins. Additionally, loss of N‐cadherin at excitatory synapses triggered increases in the density of markers for inhibitory synapses and decreased severity of hippocampal seizures. Finally, adult mutant mice were profoundly impaired in hippocampal‐dependent memory for spatial episodes. These results demonstrate a novel function for the N‐cadherin/β‐catenin complex in regulating ionotropic receptor composition of excitatory synapses, an appropriate balance of excitatory and inhibitory synaptic proteins and the maintenance of neural circuitry necessary to generate flexible yet persistent cognitive and synaptic function. © 2014 Wiley Periodicals, Inc.