Nanoscale organization of Nicastrin, the substrate receptor of the γ-secretase complex, as independent molecular domains.

Alterations in the canonical processing of Amyloid Precursor Protein generate proteoforms that contribute to the onset of Alzheimer's Disease. Modified composition of γ-secretase or mutations in its subunits has been directly linked to altered generation of Amyloid beta. Despite biochemical evidence about the role of γ-secretase in the generation of APP, the molecular origin of how spatial heterogeneity in the generation of proteoforms arises is not well understood. Here, we evaluated the localization of Nicastrin, a γ-secretase subunit, at nanometer sized functional zones of the synapse. With the help of super resolution microscopy, we confirm that Nicastrin is organized into nanodomains of high molecular density within an excitatory synapse. A similar nanoorganization was also observed for APP and the catalytic subunit of γ-secretase, Presenilin 1, that were discretely associated with Nicastrin nanodomains. Though Nicastrin is a functional subunit of γ-secretase, the Nicastrin and Presenilin1 nanodomains were either colocalized or localized independent of each other. The Nicastrin and Presenilin domains highlight a potential independent regulation of these molecules different from their canonical secretase function. The collisions between secretases and substrate molecules decide the probability and rate of product formation for transmembrane proteolysis. Our observations of secretase nanodomains indicate a spatial difference in the confinement of substrate and secretases, affecting the local probability of product formation by increasing their molecular availability, resulting in differential generation of proteoforms even within single synapses.

Rapid Ultrastructural Changes in the PSD and Surrounding Membrane after Induction of Structural LTP in Single Dendritic Spines.

The structural plasticity of dendritic spines is considered to be an important basis of synaptic plasticity, learning, and memory. Here, we induced input-specific structural LTP (sLTP) in single dendritic spines in organotypic hippocampal slices from mice of either sex and performed ultrastructural analyses of the spines using efficient correlative light and electron microscopy. We observed reorganization of the PSD nanostructure, such as perforation and segmentation, at 2-3, 20, and 120 min after sLTP induction. In addition, PSD and nonsynaptic axon-spine interface (nsASI) membrane expanded unevenly during sLTP. Specifically, the PSD area showed a transient increase at 2-3 min after sLTP induction. The PSD growth was to a degree less than spine volume growth at 2-3 min and 20 min after sLTP induction but became similar at 120 min. On the other hand, the nsASI area showed a profound and lasting expansion, to a degree similar to spine volume growth throughout the process. These rapid ultrastructural changes in PSD and surrounding membrane may contribute to rapid electrophysiological plasticity during sLTP.SIGNIFICANCE STATEMENT To understand the ultrastructural changes during synaptic plasticity, it is desired to efficiently image single dendritic spines that underwent structural plasticity in electron microscopy. We induced structural long-term potentiation (sLTP) in single dendritic spines by two-photon glutamate uncaging. We then identified the same spines at different phases of sLTP and performed ultrastructural analysis by using an efficient correlative light and electron microscopy method. We found that postsynaptic density undergoes dramatic modification in its structural complexity immediately after sLTP induction. Meanwhile, the nonsynaptic axon-spine interface area shows a rapid and sustained increase throughout sLTP. Our results indicate that the uneven modification of synaptic and nonsynaptic postsynaptic membrane might contribute to rapid electrophysiological plasticity during sLTP.

A large-scale nanoscopy and biochemistry analysis of postsynaptic dendritic spines.

Dendritic spines, the postsynaptic compartments of excitatory neurotransmission, have different shapes classified from 'stubby' to 'mushroom-like'. Whereas mushroom spines are essential for adult brain function, stubby spines disappear during brain maturation. It is still unclear whether and how they differ in protein composition. To address this, we combined electron microscopy and quantitative biochemistry with super-resolution microscopy to annotate more than 47,000 spines for more than 100 synaptic targets. Surprisingly, mushroom and stubby spines have similar average protein copy numbers and topologies. However, an analysis of the correlation of each protein to the postsynaptic density mass, used as a marker of synaptic strength, showed substantially more significant results for the mushroom spines. Secretion and trafficking proteins correlated particularly poorly to the strength of stubby spines. This suggests that stubby spines are less likely to adequately respond to dynamic changes in synaptic transmission than mushroom spines, which possibly explains their loss during brain maturation.

Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Distribution of cortactin in cerebellar Purkinje cell spines.

Dendritic spines are the primary sites of excitatory transmission in the mammalian brain. Spines of cerebellar Purkinje Cells (PCs) are plastic, but they differ from forebrain spines in a number of important respects, and the mechanisms of spine plasticity differ between forebrain and cerebellum. Our previous studies indicate that in hippocampal spines cortactin-a protein that stabilizes actin branch points-resides in the spine core, avoiding the spine shell. To see whether the distribution of cortactin differs in PC spines, we examined its subcellular organization using quantitative preembedding immunoelectron microscopy. We found that cortactin was enriched in the spine shell, associated with the non-synaptic membrane, and was also situated within the postsynaptic density (PSD). This previously unrecognized distribution of cortactin within PC spines may underlie structural and functional differences in excitatory spine synapses between forebrain, and cerebellum.

Increased occurrence of pathological mitochondria in astrocytic perivascular endfoot processes and neurons of idiopathic intracranial hypertension.

Idiopathic intracranial hypertension (IIH) primarily affects fertile, overweight women, and presents with the symptoms of raised intracranial pressure. The etiology is unknown but has been thought to relate to cerebrospinal fluid disturbance or cerebral venous stenosis. We have previously found evidence that IIH is also a disease of the brain parenchyma, evidenced by alterations at the neurogliovascular interface, including astrogliosis, pathological changes in the basement membrane and pericytes, and alterations of perivascular aquaporin-4. The aim of this present electron microscopic study was to examine whether mitochondria phenotype was changed in IIH, particularly focusing on perivascular astrocytic endfeet and neurons (soma and pre- and postsynaptic terminals). Cortical brain biopsies of nine reference individuals and eight IIH patients were analyzed for subcellular distribution and phenotypical features of mitochondria using transmission electron microscopy. We found significantly increased prevalence of pathological mitochondria and reduced number of normal mitochondria in astrocytic endfeet of IIH patients. The degree of astrogliosis correlated negatively with the number of normal mitochondria in astrocytic endfoot processes. Moreover, we found significantly increased number of pathological mitochondria in pre- and postsynaptic neuronal terminals, as well as significantly shortened distance between mitochondria and endoplasmic reticulum contacts. Finally, the length of postsynaptic density, a marker of synaptic strength, was on average reduced in IIH. The present data provide evidence of pathological mitochondria in perivascular astrocytes endfeet and neurons of IIH patients, highlighting that impaired metabolism at the neurogliovascular interface may be a facet of IIH.

Neto-α Controls Synapse Organization and Homeostasis at the Drosophila Neuromuscular Junction.

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Rapid 3D Enhanced Resolution Microscopy Reveals Diversity in Dendritic Spinule Dynamics, Regulation, and Function.

Dendritic spinules are thin protrusions, formed by neuronal spines, not adequately resolved by diffraction-limited light microscopy, which has limited our understanding of their behavior. Here we performed rapid structured illumination microscopy and enhanced resolution confocal microscopy to study spatiotemporal spinule dynamics in cortical pyramidal neurons. Spinules recurred at the same locations on mushroom spine heads. Most were short-lived, dynamic, exploratory, and originated near simple PSDs, whereas a subset was long-lived, elongated, and associated with complex PSDs. These subtypes were differentially regulated by Ca2+ transients. Furthermore, the postsynaptic Rac1-GEF kalirin-7 regulated spinule formation, elongation, and recurrence. Long-lived spinules often contained PSD fragments, contacted distal presynaptic terminals, and formed secondary synapses. NMDAR activation increased spinule number, length, and contact with distal presynaptic elements. Spinule subsets, dynamics, and recurrence were validated in cortical neurons of acute brain slices. Thus, we identified unique properties, regulatory mechanisms, and functions of spinule subtypes, supporting roles in neuronal connectivity.

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Phase separation at the synapse.

Emerging evidence indicates that liquid-liquid phase separation, the formation of a condensed molecular assembly within another diluted aqueous solution, is a means for cells to organize highly condensed biological assemblies (also known as biological condensates or membraneless compartments) with very broad functions and regulatory properties in different subcellular regions. Molecular machineries dictating synaptic transmissions in both presynaptic boutons and postsynaptic densities of neuronal synapses may be such biological condensates. Here we review recent developments showing how phase separation can build dense synaptic molecular clusters, highlight unique features of such condensed clusters in the context of synaptic development and signaling, discuss how aberrant phase-separation-mediated synaptic assembly formation may contribute to dysfunctional signaling in psychiatric disorders, and present some challenges and opportunities of phase separation in synaptic biology.

Long-term Memory Upscales Volume of Postsynaptic Densities in the Process that Requires Autophosphorylation of αCaMKII.

It is generally accepted that formation and storage of memory relies on alterations of the structure and function of brain circuits. However, the structural data, which show learning-induced and long-lasting remodeling of synapses, are still very sparse. Here, we reconstruct 1927 dendritic spines and their postsynaptic densities (PSDs), representing a postsynaptic part of the glutamatergic synapse, in the hippocampal area CA1 of the mice that underwent spatial training. We observe that in young adult (5 months), mice volume of PSDs, but not the volume of the spines, is increased 26 h after the training. The training-induced growth of PSDs is specific for the dendritic spines that lack smooth endoplasmic reticulum and spine apparatuses, and requires autophosphorylation of αCaMKII. Interestingly, aging alters training-induced ultrastructural remodeling of dendritic spines. In old mice, both the median volumes of dendritic spines and PSDs shift after training toward bigger values. Overall, our data support the hypothesis that formation of memory leaves long-lasting footprint on the ultrastructure of brain circuits; however, the form of circuit remodeling changes with age.

Autism candidate gene DIP2A regulates spine morphogenesis via acetylation of cortactin.

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

Postsynaptic densities fragment into subcomplexes upon sonication.

Postsynaptic density (PSD) fractions were isolated from rat forebrain and sonicated. Pellets from sonicated samples examined by electron microscopy revealed particles with an electron density similar to PSDs that appeared to be fragments of PSDs. Immuno-gold labeling confirmed that some of these contained PSD-95 and/or SynGAP. Biochemical analysis of supernatant and pellet fractions from sonicated samples showed almost complete recovery of several major PSD components (SynGAP, PSD-95, Shank3, Homer and Glutamate receptors) in the pellet, while the supernatant contained known contaminants of PSD fractions, such as glial acidic fibrillary protein and neurofilament protein, as well as actin and α-actinin, indicating susceptibility of these cytoskeletal elements to mechanical disruption. Size distributions of particulate material in control and sonicated samples were clearly different, with particles in the 40-90 nm range observed only in sonicated samples. Fragmentation of the PSD into subcomplexes containing major constituents suggests a patchwork structure consisting of weakly bound modules, that can be readily dissociated from each other through mechanical disruption. Modular organization and weak association between modules would endow the PSD with lateral structural flexibility.

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures.

Activity can induce structural changes in glutamatergic excitatory synapses, including increase in thickness and curvature of the postsynaptic density (PSD); these structural changes can only be documented by electron microscopy. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, increases in thickness and curvature of PSDs were noticeable within 30 s of stimulation and progressed with time up to 3 min. These structural changes were reversible upon returning the samples to control medium for 5-10 min. Thus, the postsynaptic density is a very dynamic structure that undergoes rapid reorganization of its components upon stimulation, and recovery upon cessation of stimulation. The gradual increase in thickness of PSD could result from a gradual translocation of some PSD proteins to the PSD, and the increase in curvature of the PSD is likely led by postsynaptic elements.

Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Estrogen Alters the Synaptic Distribution of Phospho-GluN2B in the Dorsolateral Prefrontal Cortex While Promoting Working Memory in Aged Rhesus Monkeys.

Age- and menopause-related deficits in working memory can be partially restored with estradiol replacement in women and female nonhuman primates. Working memory is a cognitive function reliant on persistent firing of dorsolateral prefrontal cortex (dlPFC) neurons that requires the activation of GluN2B-containing glutamate NMDA receptors. We tested the hypothesis that the distribution of phospho-Tyr1472-GluN2B (pGluN2B), a predominant form of GluN2B seen at the synapse, is sensitive to aging or estradiol treatment and coupled to working memory performance. First, ovariectomized young and aged rhesus monkeys (Macaca mulatta) received long-term cyclic vehicle (V) or estradiol (E) treatment and were tested on the delayed response (DR) test of working memory. Then, serial section electron microscopic immunocytochemistry was performed to quantitatively assess the subcellular distribution of pGluN2B. While the densities of pGluN2B immunogold particles in dlPFC dendritic spines were not different across age or treatment groups, the percentage of gold particles located within the synaptic compartment was significantly lower in aged-E monkeys compared to young-E and aged-V monkeys. On the other hand, the percentage of pGluN2B gold particles in the spine cytoplasm was decreased with E treatment in young, but increased with E in aged monkeys. In aged monkeys, DR average accuracy inversely correlated with the percentage of synaptic pGluN2B, while it positively correlated with the percentage of cytoplasmic pGluN2B. Together, E replacement may promote cognitive health in aged monkeys, in part, by decreasing the relative representation of synaptic pGluN2B and potentially protecting the dlPFC from calcium toxicity.

Changes in the Synaptic Proteome in Tauopathy and Rescue of Tau-Induced Synapse Loss by C1q Antibodies.

Synapse loss and Tau pathology are hallmarks of Alzheimer's disease (AD) and other tauopathies, but how Tau pathology causes synapse loss is unclear. We used unbiased proteomic analysis of postsynaptic densities (PSDs) in Tau-P301S transgenic mice to identify Tau-dependent alterations in synapses prior to overt neurodegeneration. Multiple proteins and pathways were altered in Tau-P301S PSDs, including depletion of a set of GTPase-regulatory proteins that leads to actin cytoskeletal defects and loss of dendritic spines. Furthermore, we found striking accumulation of complement C1q in the PSDs of Tau-P301S mice and AD patients. At synapses, C1q decorated perisynaptic membranes, accumulated in correlation with phospho-Tau, and was associated with augmented microglial engulfment of synapses and decline of synapse density. A C1q-blocking antibody inhibited microglial synapse removal in cultured neurons and in Tau-P301S mice, rescuing synapse density. Thus, inhibiting complement-mediated synapse removal by microglia could be a potential therapeutic target for Tau-associated neurodegeneration.

Functional organization of postsynaptic glutamate receptors.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA- and NMDA-type ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales. These functionally distinct receptor sub-types are co-expressed at individual synapses, allowing for the precise temporal modulation of postsynaptic excitability and plasticity. Intriguingly, these receptors are differentially distributed with respect to the presynaptic release site. While iGluRs are enriched in the core of the synapse directly opposing the release site, mGluRs reside preferentially at the border of the synapse. As such, to understand the differential contribution of these receptors to synaptic transmission, it is important to not only consider their signaling properties, but also the mechanisms that control the spatial segregation of these receptor types within synapses. In this review, we will focus on the mechanisms that control the organization of glutamate receptors at the postsynaptic membrane with respect to the release site, and discuss how this organization could regulate synapse physiology.

In vivo STED microscopy visualizes PSD95 sub-structures and morphological changes over several hours in the mouse visual cortex.

The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex. Adult knock-in mice expressing eGFP fused to the endogenous PSD95 protein were imaged at time points from 1 min to 6 h. Superresolved large assemblies of PSD95 show different sub-structures; most large assemblies were ring-like, some horse-shoe or figure-8 shaped, and shapes were continuous or made up of nanoclusters. The sub-structure appeared stable during the shorter (minute) time points, but after 1 h, more than 50% of the large assemblies showed a change in sub-structure. Overall, these data showed a sub-morphology of large PSD95 assemblies which undergo changes within the 6 hours of observation in the anaesthetised mouse.

mGluR2 versus mGluR3 Metabotropic Glutamate Receptors in Primate Dorsolateral Prefrontal Cortex: Postsynaptic mGluR3 Strengthen Working Memory Networks.

The newly evolved circuits in layer III of primate dorsolateral prefrontal cortex (dlPFC) generate the neural representations that subserve working memory. These circuits are weakened by increased cAMP-K+ channel signaling, and are a focus of pathology in schizophrenia, aging, and Alzheimer's disease. Cognitive deficits in these disorders are increasingly associated with insults to mGluR3 metabotropic glutamate receptors, while reductions in mGluR2 appear protective. This has been perplexing, as mGluR3 has been considered glial receptors, and mGluR2 and mGluR3 have been thought to have similar functions, reducing glutamate transmission. We have discovered that, in addition to their astrocytic expression, mGluR3 is concentrated postsynaptically in spine synapses of layer III dlPFC, positioned to strengthen connectivity by inhibiting postsynaptic cAMP-K+ channel actions. In contrast, mGluR2 is principally presynaptic as expected, with only a minor postsynaptic component. Functionally, increase in the endogenous mGluR3 agonist, N-acetylaspartylglutamate, markedly enhanced dlPFC Delay cell firing during a working memory task via inhibition of cAMP signaling, while the mGluR2 positive allosteric modulator, BINA, produced an inverted-U dose-response on dlPFC Delay cell firing and working memory performance. These data illuminate why insults to mGluR3 would erode cognitive abilities, and support mGluR3 as a novel therapeutic target for higher cognitive disorders.