Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo.

Four modes of endocytosis and subsequent synaptic vesicle (SV) recycling have been described at the presynapse to ensure the availability of SVs for synaptic release. However, it is unclear to what extend these modes operate under physiological activity patterns in vivo. The coat protein clathrin can regenerate SVs either directly from the plasma membrane (PM) via clathrin-mediated endocytosis (CME), or indirectly from synaptic endosomes by SV budding. Here, we examined the role of clathrin in SV recycling under physiological conditions by applying the clathrin inhibitor Pitstop-2 to the calyx of Held, a synapse optimized for high frequency synaptic transmission in the auditory brainstem, in vivo. The effects of clathrin-inhibition on SV recycling were investigated by serial sectioning scanning electron microscopy (S3EM) and 3D reconstructions of endocytic structures labeled by the endocytosis marker horseradish peroxidase (HRP). We observed large endosomal compartments as well as HRP-filled, black SVs (bSVs) that have been recently recycled. The application of Pitstop-2 led to reduced bSV but not large endosome density, increased volumes of large endosomes and shifts in the localization of both types of endocytic compartments within the synapse. These changes after perturbation of clathrin function suggest that clathrin plays a role in SV recycling from both, the PM and large endosomes, under physiological activity patterns, in vivo.

Layer-specific pain relief pathways originating from primary motor cortex.

The primary motor cortex (M1) is involved in the control of voluntary movements and is extensively mapped in this capacity. Although the M1 is implicated in modulation of pain, the underlying circuitry and causal underpinnings remain elusive. We unexpectedly unraveled a connection from the M1 to the nucleus accumbens reward circuitry through a M1 layer 6-mediodorsal thalamus pathway, which specifically suppresses negative emotional valence and associated coping behaviors in neuropathic pain. By contrast, layer 5 M1 neurons connect with specific cell populations in zona incerta and periaqueductal gray to suppress sensory hypersensitivity without altering pain affect. Thus, the M1 employs distinct, layer-specific pathways to attune sensory and aversive-emotional components of neuropathic pain, which can be exploited for purposes of pain relief.

From ensembles to meta-ensembles: Specific reward encoding by correlated network activity.

Neuronal ensembles are local, sparsely distributed populations of neurons that are reliably re-activated by a specific stimulus, context or task. Such discrete cell populations can be defined either functionally, by electrophysiological recordings or in vivo calcium imaging, or anatomically, using the expression of markers such as the immediate early gene cFos. A typical example of tasks that involve the formation of neuronal ensembles is reward learning, such as the cue-reward pairing during operant conditioning. These ensembles are re-activated during cue-presentation and increasing evidence suggests that this re-activation is the neurophysiological basis for the execution of reward-seeking behavior. Whilst the pursuit of rewards is a common daily activity, it is also related to the consumption of drugs, such as alcohol, and may result in problematic behaviors including addiction. Recent research has identified neuronal ensembles in several reward-related brain regions that control distinct aspects of a conditioned response, e.g., contextual information about the availability of a specific reward or the actions needed to retrieve this reward under the given circumstances. Here, we review studies using the activity marker cFos to identify and characterize neuronal ensembles related to alcohol and non-drug rewards with a special emphasis on the discrimination between different rewards by meta-ensembles, i.e., by dynamic co-activation of multiple ensembles across different brain areas.

Loss of miR-183/96 Alters Synaptic Strength via Presynaptic and Postsynaptic Mechanisms at a Central Synapse.

A point mutation in miR-96 causes non-syndromic progressive peripheral hearing loss and alters structure and physiology of the central auditory system. To gain further insight into the functions of microRNAs (miRNAs) within the central auditory system, we investigated constitutive Mir-183/96dko mice of both sexes. In this mouse model, the genomically clustered miR-183 and miR-96 are constitutively deleted. It shows significantly and specifically reduced volumes of auditory hindbrain nuclei, because of decreases in cell number and soma size. Electrophysiological analysis of the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) demonstrated strongly altered synaptic transmission in young-adult mice. We observed an increase in quantal content and readily releasable vesicle pool size in the presynapse while the overall morphology of the calyx was unchanged. Detailed analysis of the active zones (AZs) revealed differences in its molecular composition and synaptic vesicle (SV) distribution. Postsynaptically, altered clustering and increased synaptic abundancy of the AMPA receptor subunit GluA1 was observed resulting in an increase in quantal amplitude. Together, these presynaptic and postsynaptic alterations led to a 2-fold increase of the evoked excitatory postsynaptic currents in MNTB neurons. None of these changes were observed in deaf Cldn14ko mice, confirming an on-site role of miR-183 and miR-96 in the auditory hindbrain. Our data suggest that the Mir-183/96 cluster plays a key role for proper synaptic transmission at the calyx of Held and for the development of the auditory hindbrain.SIGNIFICANCE STATEMENT The calyx of Held is the outstanding model system to study basic synaptic physiology. Yet, genetic factors driving its morphologic and functional maturation are largely unknown. Here, we identify the Mir-183/96 cluster as an important factor to regulate its synaptic strength. Presynaptically, Mir-183/96dko calyces show an increase in release-ready synaptic vesicles (SVs), quantal content and abundance of the proteins Bassoon and Piccolo. Postsynaptically, the quantal size as well as number and size of GluA1 puncta were increased. The two microRNAs (miRNAs) are thus attractive candidates for regulation of synaptic maturation and long-term adaptations to sound levels. Moreover, the different phenotypic outcomes of different types of mutations in the Mir-183 cluster corroborate the requirement of mutation-tailored therapies in patients with hearing loss.

Alcohol and sweet reward are encoded by distinct meta-ensembles.

Cue-reward associations form distinct memories that can drive appetitive behaviors and cravings for both drugs and natural rewards. It is still unclear how such memories are encoded in the brain's reward system. We trained rats to concurrently self-administer either alcohol or a sweet saccharin solution as drug or natural rewards, respectively. Memory recall due to cue exposure reactivated reward-associated functional ensembles in reward-related brain regions, marked by a neural cFos response. While the local ensembles activated by cue presentation for either reward consisted of similar numbers of neurons, using advanced statistical network theory, we found robust reward-specific co-activation patterns across brain regions. Interestingly, the resulting meta-ensemble networks differed by the most influential regions, which in case of saccharin comprised the prefrontal cortex, while for alcohol seeking control shifted to insular cortex with strong involvement of the amygdala. Our results support the view of memory representation as a differential co-activation of local neuronal ensembles. This article is part of the special issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Cerebellar and hepatic alterations in ACBD5-deficient mice are associated with unexpected, distinct alterations in cellular lipid homeostasis.

ACBD5 deficiency is a novel peroxisome disorder with a largely uncharacterized pathology. ACBD5 was recently identified in a tethering complex mediating membrane contacts between peroxisomes and the endoplasmic reticulum (ER). An ACBD5-deficient mouse was analyzed to correlate ACBD5 tethering functions with the disease phenotype. ACBD5-deficient mice exhibit elevated very long-chain fatty acid levels and a progressive cerebellar pathology. Liver did not exhibit pathologic changes but increased peroxisome abundance and drastically reduced peroxisome-ER contacts. Lipidomics of liver and cerebellum revealed tissue-specific alterations in distinct lipid classes and subspecies. In line with the neurological pathology, unusual ultra-long chain fatty acids (C > 32) were elevated in phosphocholines from cerebelli but not liver indicating an organ-specific imbalance in fatty acid degradation and elongation pathways. By contrast, ether lipid formation was perturbed in liver towards an accumulation of alkyldiacylglycerols. The alterations in several lipid classes suggest that ACBD5, in addition to its acyl-CoA binding function, might maintain peroxisome-ER contacts in order to contribute to the regulation of anabolic and catabolic cellular lipid pathways.

Glutamatergic synaptic input to glioma cells drives brain tumour progression.

A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.

Choice for Drug or Natural Reward Engages Largely Overlapping Neuronal Ensembles in the Infralimbic Prefrontal Cortex.

Cue-reward associations form distinct memories that can drive appetitive behaviors and are involved in craving for both drugs and natural rewards. Distinct sets of neurons, so-called neuronal ensembles, in the infralimbic area (IL) of the medial prefrontal cortex (mPFC) play a key role in alcohol seeking. Whether this ensemble is specific for alcohol or controls reward seeking in general remains unclear. Here, we compared IL ensembles formed upon recall of drug (alcohol) or natural reward (saccharin) memories in male Wistar rats. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal, we found that cue-induced seeking of either alcohol or saccharin activated ensembles of similar size and organization, whereby these ensembles consist of largely overlapping neuronal populations. Thus, the IL seems to act as a general integration hub for reward seeking behavior, but also contains subsets of neurons that encode for the different rewards.SIGNIFICANCE STATEMENT Cue-reward associations form distinct memories that can act as drivers of appetitive behaviors and are involved in craving for natural rewards as well as for drugs. Distinct sets of neurons, so-called neuronal ensembles, in the infralimbic area of the mPFC play a key role in cue-triggered reward seeking. However, it is unclear whether these ensembles act as broadly tuned controllers of approach behavior or represent the learned associations between specific cues and rewards. Using an experimental framework that allows identification of two distinct reward-associated ensembles within the same animal we find largely overlapping neuronal populations. Repeated activation by two distinct events could reflect the linking of the two memory traces within the same neuron.

The presynaptic scaffolding protein Piccolo organizes the readily releasable pool at the calyx of Held.

KEY POINTS: Bassoon and Piccolo do not mediate basal synaptic vesicle release at a high-frequency synapse. Knockdown of Bassoon increases short-term depression at the calyx of Held. Both Bassoon and Piccolo have shared functions in synaptic vesicle replenishment during high-frequency synaptic transmission. Piccolo organizes the readily releasable pool of synaptic vesicles. It safeguards a fraction of them to be not immediately available for action potential-induced release. This enables the synapse to sustain high-frequency synaptic transmission over long periods. ABSTRACT: Synaptic vesicles (SVs) are released at the active zone (AZ), a specialized region of the presynaptic plasma membrane organized by a highly interconnected network of multidomain proteins called the cytomatrix of the active zone (CAZ). Two core components of the CAZ are the large, highly homologous scaffolding proteins Bassoon and Piccolo, whose function is not well understood. To investigate their role in synaptic transmission, we established the small hairpin RNA (shRNA)-mediated in vivo knockdown (KD) of Bassoon and Piccolo at the rat calyx of Held synapse. KD of Bassoon and Piccolo, separately or simultaneously, did not affect basic SV release. However, short-term depression (STD) was prominently increased by the KD of Bassoon, whereas KD of Piccolo only had a minor effect. The observed alterations in STD were readily explained by reduced SV replenishment in synapses deficient in either of the proteins. Thus, the regulation of SV refilling during ongoing synaptic activity is a shared function of Bassoon and Piccolo, although Bassoon appears to be more efficient. Moreover, we observed the recruitment of slowly-releasing SVs of the readily-releasable pool (RRP), which are normally not available for action potential-induced release, during high-frequency stimulation in Piccolo-deficient calyces. Therefore, the results obtained in the present study suggest a novel and specific role for Piccolo in the organization of the subpools of the RRP.

Molecular Machines Regulating the Release Probability of Synaptic Vesicles at the Active Zone.

The fusion of synaptic vesicles (SVs) with the plasma membrane of the active zone (AZ) upon arrival of an action potential (AP) at the presynaptic compartment is a tightly regulated probabilistic process crucial for information transfer. The probability of a SV to release its transmitter content in response to an AP, termed release probability (Pr), is highly diverse both at the level of entire synapses and individual SVs at a given synapse. Differences in Pr exist between different types of synapses, between synapses of the same type, synapses originating from the same axon and even between different SV subpopulations within the same presynaptic terminal. The Pr of SVs at the AZ is set by a complex interplay of different presynaptic properties including the availability of release-ready SVs, the location of the SVs relative to the voltage-gated calcium channels (VGCCs) at the AZ, the magnitude of calcium influx upon arrival of the AP, the buffering of calcium ions as well as the identity and sensitivity of the calcium sensor. These properties are not only interconnected, but can also be regulated dynamically to match the requirements of activity patterns mediated by the synapse. Here, we review recent advances in identifying molecules and molecular machines taking part in the determination of vesicular Pr at the AZ.

Modulation of Presynaptic Release Probability by the Vertebrate-Specific Protein Mover.

Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission.

An EGF receptor targeting Ranpirnase-diabody fusion protein mediates potent antitumour activity in vitro and in vivo.

Cytotoxic ribonucleases such as the leopard frog derivative Ranpirnase (Onconase(®)) have emerged as a valuable new class of cancer therapeutics. Clinical trials employing single agent Ranpirnase in cancer patients have demonstrated significant clinical activity and surprisingly low immunogenicity. However, dose-limiting toxicity due to unspecific uptake of the RNase into non-cancerous cells is reached at relatively low concentrations of > 1 mg/m(2). We have in the present study generated a dimeric anti-EGFR Ranpirnase-diabody fusion protein capable to deliver two Ranpirnase moieties per molecule to EGFR-positive tumour cells. We show that this compound mediated far superior efficacy for killing EGFR-positive tumour cells than a monomeric counterpart. Most importantly, cell killing was restricted to EGFR-positive target cells and no dose-limiting toxicity of Ranpirnase-diabody was observed in mice. These data indicate that by targeted delivery of Ranpirnase non-selective toxicity can be abolished and suggests Ranpirnase-diabody as a promising new drug for therapeutic interventions in EGFR-positive cancers.

Gene expression profile during functional maturation of a central mammalian synapse.

Calyx of Held giant presynaptic terminals in the auditory brainstem form glutamatergic axosomatic synapses that have advanced to one of the best-studied synaptic connections of the mammalian brain. As the auditory system matures and adjusts to high-fidelity synaptic transmission, the calyx undergoes extensive structural and functional changes - in mice, it is formed at about postnatal day 3 (P3), achieves immature function until hearing onset at about P10 and can be considered mature from P21 onwards. This setting provides a unique opportunity to examine the repertoire of genes driving synaptic structure and function during postnatal maturation. Here, we determined the gene expression profile of globular bushy cells (GBCs), neurons giving rise to the calyx of Held, at different maturational stages (P3, P8, P21). GBCs were retrogradely labelled by stereotaxic injection of fluorescent cholera toxin-B, and their mRNA content was collected by laser microdissection. Microarray profiling, successfully validated with real time quantitative polymerase chain reaction and nCounter approaches, revealed genes regulated during maturation. We found that mostly genes implicated in the general cell biology of the neuron were regulated, while most genes related to synaptic function were regulated around the onset of hearing. Among these, voltage-gated ion channels and calcium-binding proteins were strongly regulated, whereas most genes involved in the synaptic vesicle cycle were only moderately regulated. These results suggest that changes in the expression patterns of ion channels and calcium-binding proteins are a dominant factor in defining key synaptic properties during maturation of the calyx of Held.

Undifferentiated embryonic stem cells express ionotropic glutamate receptor mRNAs.

Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.

Endocytic structures and synaptic vesicle recycling at a central synapse in awake rats.

The synaptic vesicle (SV) cycle has been studied extensively in cultured cells and slice preparations, but not much is known about the roles and relative contributions of endocytic pathways and mechanisms of SV recycling in vivo, under physiological patterns of activity. We employed horseradish peroxidase (HRP) as an in vivo marker of endocytosis at the calyx of Held synapse in the awake rat. Ex vivo serial section scanning electron microscopy and 3D reconstructions revealed two categories of labelled structures: HRP-filled SVs and large cisternal endosomes. Inhibition of adaptor protein complexes 1 and 3 (AP-1, AP-3) by in vivo application of Brefeldin A (BFA) disrupted endosomal SV budding while SV recycling via clathrin-mediated endocytosis (CME) remained unaffected. In conclusion, our study establishes cisternal endosomes as an intermediate of the SV cycle and reveals CME and endosomal budding as the predominant mechanisms of SV recycling in a tonically active central synapse in vivo.

Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons.

Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues.

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

A small pool of vesicles maintains synaptic activity in vivo.

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈ 5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.

Self-limited oxygen exchange kinetics at SnO2 surfaces.

The oxygen exchange at SnO(2) surfaces strongly depends on surface termination, which is affected by the oxygen chemical potential. At low oxygen chemical potential, the surface adopts its reduced termination which allows oxygen exchange, while exchange is suppressed by the stoichiometric surface termination.

Oligomerization in the endoplasmic reticulum and intracellular trafficking of kainate receptors are subunit-dependent but not editing-dependent.

Investigating subunit assembly of ionotropic glutamate receptor complexes and their trafficking to the plasma membrane under physiological conditions in live cells has been challenging. By confocal imaging of fluorescently labeled kainate receptor (KAR) subunits combined with digital co-localization and fluorescence resonance energy (FRET) transfer analyses, we investigated the assembly of homomeric and heteromeric receptor complexes and identified the subcellular location of subunit interactions. Our data provide direct evidence for oligomerization of KAR subunits as early as following their biosynthesis in the endoplasmic reticulum (ER). These oligomeric assemblies pass through the Golgi apparatus en route to the plasma membrane. We show that the amino acid at the Q/R editing site of the KAR subunit GluR6 neither determines subunit oligomerization in the ER nor ER exit or plasma membrane expression, and that it does not alter GluR6 interaction with KA2. This finding sets KARs apart from alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, where in the absence of auxiliary proteins Q isoforms exit the ER much more efficiently than R isoforms. Furthermore, although KA2 subunits do not form functional homotetrameric complexes, we visualized their oligomerization (at least dimerization) in the ER. Finally, we demonstrate that plasma membrane expression of GluR6/KA2 heteromeric complexes is modulated not only by GluR6 but also KA2.