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P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Cav2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ~40 nm. Calcium nanodomain created by the opening of Cav2.1 channels likely activates the two channels that limit the extent of depolarization. [ABSTRACT FROM AUTHOR]

The time course of signaling via heterotrimeric G proteins is controlled through their activation by G-protein coupled receptors and deactivation through the action of GTPase accelerating proteins (GAPs). Here we identify RGS7 and RGS11 as the key GAPs in the mGluR6 pathway of retinal rod ON bipolar cells that set the sensitivity and time course of light-evoked responses. We showed using electroretinography and single cell recordings that the elimination of RGS7 did not influence dark-adapted light-evoked responses, but the concurrent elimination of RGS11 severely reduced their magnitude and dramatically slowed the onset of the response. In RGS7/RGS11 double-knockout mice, light-evoked responses in rod ON bipolar cells were only observed during persistent activation of rod photoreceptors that saturate rods. These observations are consistent with persistently high G-protein activity in rod ON bipolar cell dendrites caused by the absence of the dominant GAP, biasing TRPM1 channels to the closed state. [ABSTRACT FROM AUTHOR]

Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90—100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36. [ABSTRACT FROM AUTHOR]

In an n-alkane-assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2.3.1.7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489–492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The aniino-terniinal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalls cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondrial CATs arose from the difference in the initiation sites of translation. [ABSTRACT FROM AUTHOR]

The peroxisomal isocitrate lyase (ICL) of an n-alkane-utilizable yeast, Candida tropicalis, could be transported into the peroxisomes of Saccharomyces cerevisiae MT8-1 cells grown in an oleic acid medium, even if strains were different. To identify the important regions to direct C. tropicalis ICL (CT-ICL) to its proper subcellular location in S. cerevisiae, we observed the subcellular localization of the expressed products of mutant CT-ICL genes in S. cerevisiae by immunoelectron microscopy. Under the control of the UPR-ICL promoter, which enhanced gene expression by proliferation of the peroxisomes, the following mutant CT-ICL genes were constructed and expressed: (i) truncated or mutant genes in the C-terminal region which apparently contained peroxisomal targeting signals, Δ550, Δ549–550, Δ548–550, Δ548–550 + SKL and Δ340–550; and (ii) four internal deletion mutant genes, Δ41–115, Δ41–239, Δ41–325 and Δ237–339. The results showed that three C-terminal amino acid residues were not essential for targeting the peroxisomes in this enzyme. In cells harboring the deleted internal regions of mutant ICL genes, the expressed polypeptides did not target the peroxisomes and the polypeptides, except for Δ41–115, were mistargeted to mitochondria. We observed an unknown structure which we called a “protein aggregate body (PAB),” in which various mutant CT-ICL polypeptides aggregated. [ABSTRACT FROM PUBLISHER]

The time course of BMY-28864-dependent morphological changes in A9540 was studied by electron microscopy. Scanning electron microscopy revealed that BMY-28864 often induced cell surface deformations such as abnormal swelling and bulging around budding sites and bud scars. The cell membrane damage was visualized by freeze-fracturing technique as deep pit-like invaginations. Transmission electron microscopy with thin-sectioned specimens also demonstrated that BMY-28864 induced cell membrane invaginations together with cell membrane detachment from the cell wall, nuclear membrane fragmentation and mitochondrial aberration. Statistical sequence analysis of the prominent BMY-28864-dependent morphological changes of the yeast cells led to the conclusion that BMY-28864 first attacked the cell membrane and then caused disintegration of other intracytoplasmic organelles, resulting in the lethal effect. [ABSTRACT FROM PUBLISHER]

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid β-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal β-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal β-oxidation system. [Copyright &y& Elsevier]

Summary In axons, an action potential (AP) is thought to be broadcast as an unwavering binary pulse over its arbor, driving neurotransmission uniformly at release sites. Yet by recording from axons of cerebellar stellate cell (SC) interneurons, we show that AP width varies between presynaptic bouton sites, even within the same axon branch. The varicose geometry of SC boutons alone does not impose differences in spike duration. Rather, axonal patching revealed heterogeneous peak conductance densities of currents mediated mainly by fast-activating K v 3-type potassium channels, with clustered hotspots at boutons and restricted expression at adjoining shafts. Blockade of K v channels at individual boutons indicates that currents immediately local to a release site direct spike repolarization at that location. Thus, the clustered arrangement and variable expression density of K v 3 channels at boutons are key determinants underlying compartmentalized control of AP width in a near synapse-by-synapse manner, multiplying the signaling capacity of these structures. [ABSTRACT FROM AUTHOR]

Summary In the retina, rod and cone photoreceptors form distinct connections with different classes of downstream bipolar cells. However, the molecular mechanisms responsible for their selective connectivity are unknown. Here we identify a cell-adhesion protein, ELFN1, to be essential for the formation of synapses between rods and rod ON-bipolar cells in the primary rod pathway. ELFN1 is expressed selectively in rods where it is targeted to the axonal terminals by the synaptic release machinery. At the synapse, ELFN1 binds in trans to mGluR6, the postsynaptic receptor on rod ON-bipolar cells. Elimination of ELFN1 in mice prevents the formation of synaptic contacts involving rods, but not cones, allowing a dissection of the contributions of primary and secondary rod pathways to retinal circuit function and vision. We conclude that ELFN1 is necessary for the selective wiring of rods into the primary rod pathway and is required for high sensitivity of vision. [ABSTRACT FROM AUTHOR]

The Author Contributions section now reads: "Conceptualization: D.J., J.M.C., N.K., W.E.H., and M.S.; Investigation: D.J., E.S., K.S., and D.G-G.; Writing and Editing, D.J., E.S., D.G-G., J.M.C., N.K., W.E.H., and M.S.; Funding Acquisition: J.M.C.; Supervision: J.M.C, N.K., W.E.H., and M.S." In addition, Diego Jerez and Eleanor Stuart were incorrectly affiliated with 'Florida Atlantic University, 777 Glades Rd, Boca Raton, FL, 33431, USA'. The original article can be found online at https://doi.org/10.1038/s41598-021-87015-2. Correction to: I Scientific Reports i https://doi.org/10.1038/s41598-021-87015-2, published online 08 April 2021 William E. Hahn was omitted from the author list in the original version of this Article. [Extracted from the article]

Summary Neural circuit wiring relies on selective synapse formation whereby a presynaptic release apparatus is matched with its cognate postsynaptic machinery. At metabotropic synapses, the molecular mechanisms underlying this process are poorly understood. In the mammalian retina, rod photoreceptors form selective contacts with rod ON-bipolar cells by aligning the presynaptic voltage-gated Ca 2+ channel directing glutamate release (Ca V 1.4) with postsynaptic mGluR6 receptors. We show this coordination requires an extracellular protein, α2δ4, which complexes with Ca V 1.4 and the rod synaptogenic mediator, ELFN1, for trans-synaptic alignment with mGluR6. Eliminating α2δ4 in mice abolishes rod synaptogenesis and synaptic transmission to rod ON-bipolar cells, and disrupts postsynaptic mGluR6 clustering. We further find that in rods, α2δ4 is crucial for organizing synaptic ribbons and setting Ca V 1.4 voltage sensitivity. In cones, α2δ4 is essential for Ca V 1.4 function, but is not required for ribbon organization, synaptogenesis, or synaptic transmission. These findings offer insights into retinal pathologies associated with α2δ4 dysfunction. [ABSTRACT FROM AUTHOR]

Most excitatory synapses in the mammalian brain are contacted or ensheathed by astrocyte processes, forming tripartite synapses. Astrocytes are thought to be critical regulators of the structural and functional dynamics of synapses. While the degree of synaptic coverage by astrocytes is known to vary across brain regions and animal species, the reason for and implications of this variability remains unknown. Further, how astrocyte coverage of synapses relates to in vivo functional properties of individual synapses has not been investigated. Here, we characterized astrocyte coverage of synapses of pyramidal neurons in the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and sensory-evoked Ca 2+ activity. Nearly, all synapses were contacted by astrocytes, and most were contacted along the axon-spine interface. Structurally, we found that the degree of synaptic astrocyte coverage directly scaled with synapse size and postsynaptic density complexity. Functionally, we found that the amount of astrocyte coverage scaled with how selectively a synapse responds to a particular visual stimulus and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca 2+ events. Our study shows astrocyte coverage is highly correlated with structural metrics of synaptic strength of excitatory synapses in the visual cortex and demonstrates a previously unknown relationship between astrocyte coverage and reliable sensory activation., (© 2024 The Author(s). GLIA published by Wiley Periodicals LLC.)

Postsynaptic mitochondria are critical for the development, plasticity, and maintenance of synaptic inputs. However, their relationship to synaptic structure and functional activity is unknown. We examined a correlative dataset from ferret visual cortex with in vivo two-photon calcium imaging of dendritic spines during visual stimulation and electron microscopy reconstructions of spine ultrastructure, investigating mitochondrial abundance near functionally and structurally characterized spines. Surprisingly, we found no correlation to structural measures of synaptic strength. Instead, we found that mitochondria are positioned near spines with orientation preferences that are dissimilar to the somatic preference. Additionally, we found that mitochondria are positioned near groups of spines with heterogeneous orientation preferences. For a subset of spines with a mitochondrion in the head or neck, synapses were larger and exhibited greater selectivity to visual stimuli than those without a mitochondrion. Our data suggest mitochondria are not necessarily positioned to support the energy needs of strong spines, but rather support the structurally and functionally diverse inputs innervating the basal dendrites of cortical neurons., Competing Interests: CT, MR, NK, BS No competing interests declared, (© 2023, Thomas et al.)

The formation of neural circuits occurs in a programmed fashion, but proper activity in the circuit is essential for refining the organization necessary for driving complex behavioral tasks. In the retina, sensory deprivation during the critical period of development is well known to perturb the organization of the visual circuit making the animals unable to use vision for behavior. However, the extent of plasticity, molecular factors involved, and malleability of individual channels in the circuit to manipulations outside of the critical period are not well understood. In this study, we selectively disconnected and reconnected rod photoreceptors in mature animals after completion of the retina circuit development. We found that introducing synaptic rod photoreceptor input post-developmentally allowed their integration into the circuit both anatomically and functionally. Remarkably, adult mice with newly integrated rod photoreceptors gained high-sensitivity vision, even when it was absent from birth. These observations reveal plasticity of the retina circuit organization after closure of the critical period and encourage the development of vision restoration strategies for congenital blinding disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1's roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability., Competing Interests: CK, MA, TR, CT, PV, DG, MR, HT, NK, SY No competing interests declared, (© 2022, Keine et al.)

Electron microscopy (EM) enables high-resolution visualization of protein distributions in biological tissues. For detection, gold nanoparticles are typically used as an electron-dense marker for immunohistochemically labeled proteins. Manual annotation of gold particle labels is laborious and time consuming, as gold particle counts can exceed 100,000 across hundreds of image segments to obtain conclusive data sets. To automate this process, we developed Gold Digger, a software tool that uses a modified pix2pix deep learning network capable of detecting and annotating colloidal gold particles in biological EM images obtained from both freeze-fracture replicas and plastic sections prepared with the post-embedding method. Gold Digger performs at near-human-level accuracy, can handle large images, and includes a user-friendly tool with a graphical interface for proof reading outputs by users. Manual error correction also helps for continued re-training of the network to improve annotation accuracy over time. Gold Digger thus enables rapid high-throughput analysis of immunogold-labeled EM data and is freely available to the research community.

Single neocortical neurons are driven by populations of excitatory inputs, which form the basis of neuronal selectivity to features of sensory input. Excitatory connections are thought to mature during development through activity-dependent Hebbian plasticity 1 , whereby similarity between presynaptic and postsynaptic activity selectively strengthens some synapses and weakens others 2 . Evidence in support of this process includes measurements of synaptic ultrastructure and in vitro and in vivo physiology and imaging studies 3-8 . These corroborating lines of evidence lead to the prediction that a small number of strong synaptic inputs drive neuronal selectivity, whereas weak synaptic inputs are less correlated with the somatic output and modulate activity overall 6,7 . Supporting evidence from cortical circuits, however, has been limited to measurements of neighbouring, connected cell pairs, raising the question of whether this prediction holds for a broad range of synapses converging onto cortical neurons. Here we measure the strengths of functionally characterized excitatory inputs contacting single pyramidal neurons in ferret primary visual cortex (V1) by combining in vivo two-photon synaptic imaging and post hoc electron microscopy. Using electron microscopy reconstruction of individual synapses as a metric of strength, we find no evidence that strong synapses have a predominant role in the selectivity of cortical neuron responses to visual stimuli. Instead, selectivity appears to arise from the total number of synapses activated by different stimuli. Moreover, spatial clustering of co-active inputs appears to be reserved for weaker synapses, enhancing the contribution of weak synapses to somatic responses. Our results challenge the role of Hebbian mechanisms in shaping neuronal selectivity in cortical circuits, and suggest that selectivity reflects the co-activation of large populations of presynaptic neurons with similar properties and a mixture of strengths.

Establishment of functional synaptic connections in a selective manner is essential for nervous system operation. In mammalian retinas, rod and cone photoreceptors form selective synaptic connections with different classes of bipolar cells (BCs) to propagate light signals. While there has been progress in elucidating rod wiring, molecular mechanisms used by cones to establish functional synapses with BCs have remained unknown. Using an unbiased proteomic strategy in cone-dominant species, we identified the cell-adhesion molecule ELFN2 to be pivotal for the functional wiring of cones with the ON type of BC. It is selectively expressed in cones and transsynaptically recruits the key neurotransmitter receptor mGluR6 in ON-BCs to enable synaptic transmission. Remarkably, ELFN2 in cone terminals functions in synergy with a related adhesion molecule, ELFN1, and their concerted interplay during development specifies selective wiring and transmission of cone signals. These findings identify a synaptic connectivity mechanism of cones and illustrate how interplay between adhesion molecules and postsynaptic transmitter receptors orchestrates functional synaptic specification in a neural circuit., Competing Interests: The authors declare no competing interest.

In the presynaptic terminal, the magnitude and location of Ca 2+ entry through voltage-gated Ca 2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a Ca V 2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the Ca V 2.1 current density with concomitant reductions in Ca V 2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca 2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of Ca V 2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cone photoreceptors scale dynamically the sensitivity of responses to maintain responsiveness across wide range of changes in luminance. Synaptic changes contribute to this adaptation, but how this process is coordinated at the molecular level is poorly understood. Here, we report that a cell adhesion-like molecule, LRIT1, is enriched selectively at cone photoreceptor synapses where it engages in a trans-synaptic interaction with mGluR6, the principal receptor in postsynaptic ON-bipolar cells. The levels of LRIT1 are regulated by the neurotransmitter release apparatus that controls photoreceptor output. Knockout of LRIT1 in mice increases the sensitivity of cone synaptic signaling while impairing its ability to adapt to background light without overtly influencing the morphology or molecular composition of photoreceptor synapses. Accordingly, mice lacking LRIT1 show visual deficits under conditions requiring temporally challenging discrimination of visual signals in steady background light. These observations reveal molecular mechanisms involved in scaling synaptic communication in the retina., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target., Competing Interests: The authors declare no conflict of interest.

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by Ca V 2.1 calcium channels (Ca V 2.1) and is highly dependent on the physical distance between Ca V 2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of Ca V 2.1 through direct interactions with the Ca V 2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant Ca V 2.1 α 1 subunits on a Ca V 2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of Ca V 2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for Ca V 2.1 abundance and coupling. Therefore, our work advances our molecular understanding of Ca V 2.1 regulation of neurotransmitter release in mammalian CNS synapses.

P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ∼40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.