TY - JOUR AB - The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation. AU - Bhandari, Pradeep AU - Vandael, David H AU - Fernández-Fernández, Diego AU - Fritzius, Thorsten AU - Kleindienst, David AU - Önal, Hüseyin C AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Gassmann, Martin AU - Jonas, Peter M AU - Kulik, Akos AU - Bettler, Bernhard AU - Shigemoto, Ryuichi AU - Koppensteiner, Peter ID - 9437 JF - eLife TI - GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals VL - 10 ER - TY - THES AB - Left-right asymmetries can be considered a fundamental organizational principle of the vertebrate central nervous system. The hippocampal CA3-CA1 pyramidal cell synaptic connection shows an input-side dependent asymmetry where the hemispheric location of the presynaptic CA3 neuron determines the synaptic properties. Left-input synapses terminating on apical dendrites in stratum radiatum have a higher density of NMDA receptor subunit GluN2B, a lower density of AMPA receptor subunit GluA1 and smaller areas with less often perforated PSDs. On the other hand, left-input synapses terminating on basal dendrites in stratum oriens have lower GluN2B densities than right-input ones. Apical and basal synapses further employ different signaling pathways involved in LTP. SDS-digested freeze-fracture replica labeling can visualize synaptic membrane proteins with high sensitivity and resolution, and has been used to reveal the asymmetry at the electron microscopic level. However, it requires time-consuming manual demarcation of the synaptic surface for quantitative measurements. To facilitate the analysis of replica labeling, I first developed a software named Darea, which utilizes deep-learning to automatize this demarcation. With Darea I characterized the synaptic distribution of NMDA and AMPA receptors as well as the voltage-gated Ca2+ channels in CA1 stratum radiatum and oriens. Second, I explored the role of GluN2B and its carboxy-terminus in the establishment of input-side dependent hippocampal asymmetry. In conditional knock-out mice lacking GluN2B expression in CA1 and GluN2B-2A swap mice, where GluN2B carboxy-terminus was exchanged to that of GluN2A, no significant asymmetries of GluN2B, GluA1 and PSD area were detected. We further discovered a previously unknown functional asymmetry of GluN2A, which was also lost in the swap mouse. These results demonstrate that GluN2B carboxy-terminus plays a critical role in normal formation of input-side dependent asymmetry. AU - Kleindienst, David ID - 9562 SN - 2663-337X TI - 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning ER - TY - CHAP AB - High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms. AU - Kaufmann, Walter AU - Kleindienst, David AU - Harada, Harumi AU - Shigemoto, Ryuichi ID - 9756 KW - Freeze-fracture replica: Deep learning KW - Immunogold labeling KW - Integral membrane protein KW - Electron microscopy SN - 9781071615218 T2 - Receptor and Ion Channel Detection in the Brain TI - High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL) VL - 169 ER - TY - JOUR AB - In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD‐95‐expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS‐digested freeze‐fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular–anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions. AU - Nakamoto, Chihiro AU - Konno, Kohtarou AU - Miyazaki, Taisuke AU - Nakatsukasa, Ena AU - Natsume, Rie AU - Abe, Manabu AU - Kawamura, Meiko AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi AU - Yamasaki, Miwako AU - Sakimura, Kenji AU - Watanabe, Masahiko ID - 7148 IS - 6 JF - Journal of Comparative Neurology SN - 0021-9967 TI - Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain VL - 528 ER - TY - JOUR AB - Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission. AU - Piriya Ananda Babu, Lashmi AU - Wang, Han Ying AU - Eguchi, Kohgaku AU - Guillaud, Laurent AU - Takahashi, Tomoyuki ID - 7339 IS - 1 JF - Journal of neuroscience TI - Microtubule and actin differentially regulate synaptic vesicle cycling to maintain high-frequency neurotransmission VL - 40 ER - TY - JOUR AB - Metabotropic γ-aminobutyric acid (GABAB) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABAB receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABAB1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABAB receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABAB receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABAB1 at cytoplasmic sites. GABAB receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABAB receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABAB receptors in the DG suggest the alteration of the GABAB-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD AU - Martín-Belmonte, Alejandro AU - Aguado, Carolina AU - Alfaro-Ruíz, Rocío AU - Moreno-Martínez, Ana Esther AU - De La Ossa, Luis AU - Martínez-Hernández, José AU - Buisson, Alain AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo AU - Luján, Rafael ID - 7664 IS - 7 JF - International journal of molecular sciences TI - Density of GABAB receptors is reduced in granule cells of the hippocampus in a mouse model of Alzheimer's disease VL - 21 ER - TY - JOUR AB - Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions. AU - Eguchi, Kohgaku AU - Velicky, Philipp AU - Hollergschwandtner, Elena AU - Itakura, Makoto AU - Fukazawa, Yugo AU - Danzl, Johann G AU - Shigemoto, Ryuichi ID - 7665 JF - Frontiers in Cellular Neuroscience SN - 16625102 TI - Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions VL - 14 ER - TY - JOUR AB - Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions. AU - Bao, Jin AU - Graupner, Michael AU - Astorga, Guadalupe AU - Collin, Thibault AU - Jalil, Abdelali AU - Indriati, Dwi Wahyu AU - Bradley, Jonathan AU - Shigemoto, Ryuichi AU - Llano, Isabel ID - 7878 JF - eLife TI - Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo VL - 9 ER - TY - JOUR AB - Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses. AU - Wang, Han Ying AU - Eguchi, Kohgaku AU - Yamashita, Takayuki AU - Takahashi, Tomoyuki ID - 7908 IS - 21 JF - Journal of Neuroscience TI - Frequency-dependent block of excitatory neurotransmission by isoflurane via dual presynaptic mechanisms VL - 40 ER - TY - JOUR AB - The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high‐resolution immunoelectron microscopic methods for GABAB receptors, this study provides a quantitative description of the expression and the subcellular localization of GABAB1 in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABAB1 were similar in APP/PS1 mice and in age‐matched wild‐type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABAB1 subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum‐moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABAB1 was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane‐targeted GABAB receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment‐ and age‐dependent reduction of plasma membrane‐targeted GABAB receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABAB‐mediated synaptic transmission taking place in AD. AU - Martín-Belmonte, Alejandro AU - Aguado, Carolina AU - Alfaro-Ruíz, Rocío AU - Moreno-Martínez, Ana Esther AU - De La Ossa, Luis AU - Martínez-Hernández, José AU - Buisson, Alain AU - Früh, Simon AU - Bettler, Bernhard AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo AU - Luján, Rafael ID - 7207 IS - 3 JF - Brain Pathology SN - 10156305 TI - Reduction in the neuronal surface of post and presynaptic GABA>B< receptors in the hippocampus in a mouse model of Alzheimer's disease VL - 30 ER -