Structural determinants underlying the high efficacy of synaptic transmission and plasticity at synaptic boutons in layer 4 of the adult rat 'barrel cortex'

Excitatory layer 4 (L4) neurons in the ‘barrel field’ of the rat somatosensory cortex represent an important component in thalamocortical information processing. However, no detailed information exists concerning the quantitative geometry of synaptic boutons terminating on these neurons. Thus, L4 synaptic boutons were investigated using serial ultrathin sections and subsequent quantitative 3D reconstructions. In particular, parameters representing structural correlates of synaptic transmission and plasticity such as the number, size and distribution of pre- and postsynaptic densities forming the active zone (AZ) and of the three functionally defined pools of synaptic vesicles were analyzed. L4 synaptic boutons varied substantially in shape and size; the majority had a single, but large AZ with opposing pre- and postsynaptic densities that matched perfectly in size and position. More than a third of the examined boutons showed perforations of the postsynaptic density. Synaptic boutons contained on average a total pool of 561 ± 108 vesicles, with ~5 % constituting the putative readily releasable, ~23 % the recycling, and the remainder the reserve pool. These pools are comparably larger than other characterized central synapses. Synaptic complexes were surrounded by a dense network of fine astrocytic processes that reached as far as the synaptic cleft, thus regulating the temporal and spatial glutamate concentration, and thereby shaping the unitary EPSP amplitude. In summary, the geometry and size of AZs, the comparably large readily releasable and recycling pools, together with the tight astrocytic ensheathment, may explain and contribute to the high release probability, efficacy and modulation of synaptic transmission at excitatory L4 synaptic boutons. Moreover, the structural variability as indicated by the geometry of L4 synaptic boutons, the presence of mitochondria and the size and shape of the AZs strongly suggest that synaptic reliability, strength and plasticity is governed and modulated individually at excitatory L4 synaptic boutons

Layer-specific distribution and expression pattern of AMPA- and NMDA-type glutamate receptors in the barrel field of the adult rat somatosensory cortex:a quantitative electron microscopic analysis

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-d-aspartate) glutamate receptors are driving forces for synaptic transmission and plasticity at neocortical synapses. However, their distribution pattern in the adult rat neocortex is largely unknown and was quantified using freeze fracture replication combined with postimmunogold-labeling. Both receptors were co-localized at layer (L)4 and L5 postsynaptic densities (PSDs). At L4 dendritic shaft and spine PSDs, the number of gold grains detecting AMPA was similar, whereas at L5 shaft PSDs AMPA-receptors outnumbered those on spine PSDs. Their number was significantly higher at L5 vs. L4 PSDs. At L4 and L5 dendritic shaft PSDs, the number of gold grains detecting GluN1 was ~2-fold higher than at spine PSDs. The number of gold grains detecting the GluN1-subunit was higher for both shaft and spine PSDs in L5 vs. L4. Both receptors showed a large variability in L4 and L5. A high correlation between the number of gold grains and PSD size for both receptors and targets was observed. Both receptors were distributed over the entire PSD but showed a layer- and target-specific distribution pattern.The layer- and target-specific distribution of AMPA and GluN1 glutamate receptors partially contribute to the observed functional differences in synaptic transmission and plasticity in the neocortex

Presynaptic localization of GluK5 in rod photoreceptors suggests a novel function of high affinity glutamate receptors in the mammalian retina.

Kainate receptors mediate glutamatergic signaling through both pre- and presynaptic receptors. Here, we studied the expression of the high affinity kainate receptor GluK5 in the mouse retina. Double-immunofluoresence labeling and electron microscopic analysis revealed a presynaptic localization of GluK5 in the outer plexiform layer. Unexpectedly, we found GluK5 almost exclusively localized to the presynaptic ribbon of photoreceptor terminals. Moreover, in GluK5-deficient mutant mice the structural integrity of synaptic ribbons was severely altered pointing to a novel function of GluK5 in organizing synaptic ribbons in the presynaptic terminals of rod photoreceptors

Morphology, input–output relations and synaptic connectivity of Cajal–Retzius cells in layer 1 of the developing neocortex of CXCR4-EGFP mice

Layer 1 (L1) neurons, in particular Cajal–Retzius (CR) cells are among the earliest generated neurons in the neocortex. However, their role and that of L1 GABAergic interneurons in the establishment of an early cortical microcircuit are still poorly understood. Thus, the morphology of whole-cell recorded and biocytin-filled CR cells was investigated in postnatal day (P) 7–11 old CXCR4-EGFP mice where CR cells can be easily identified by their fluorescent appearance. Confocal-, light- and subsequent electron microscopy was performed to investigate their developmental regulation, morphology, synaptic input–output relationships and electrophysiological properties. CR cells reached their peak in occurrence between P4 to P7 and from thereon declined to almost complete disappearance at P14 by undergoing selective cell death through apoptosis. CR cells formed a dense and long-range horizontal network in layer 1 with a remarkable high density of synaptic boutons along their axons. They received dense GABAergic and non-GABAergic synaptic input and in turn provided synaptic output preferentially with spines or shafts of terminal tuft dendrites of pyramidal neurons. Interestingly, no dye-coupling between CR cells with other cortical neurons was observed as reported for other species, however, biocytin-labeling of individual CR cells leads to co-staining of L1 end foot astrocytes. Electrophysiologically, CR cells are characterized by a high input resistance and a characteristic firing pattern. Increasing depolarizing currents lead to action potential of decreasing amplitude and increasing half width, often terminated by a depolarization block. The presence of membrane excitability, the high density of CR cells in layer 1, their long-range horizontal axonal projection together with a high density of synaptic boutons and their synaptic input–output relationship suggest that they are an integral part of an early cortical network important not only in layer 1 but also for the establishment and formation of the cortical column

Electron microscopic analysis of synapses deficient in GluK5.

<p>(A₁–A₈) Electron micrographs showing a series of ultrathin sections taken through two rod photoreceptor synaptic terminals of a GluK5^-/- retina. The ultrastructural abnormalities in the course of the synaptic ribbons are clearly visible. In most sections the ribbons appear disintegrated (see arrowheads in A). (B₁-B₇) Electron micrographs showing a series of ultrathin sections taken through a rod photoreceptor synaptic terminal of a GluK5 knockout mouse. The ribbon and its fragments are visualized in green. For a better visualization the postsynaptic elements are colored in light and dark blue and the outline of the rod terminal is labelled in red. (C) Overlay of the colored green ribbon fragments. It is possible to follow the course of the ribbon through the matrix of the rod terminal. The overlay suggests that although the ribbon course is undulated its continuity might be conserved. (D and E) Immunohistochemistry for CtBP2 in wild-type and GluK5^-/- retinae. (D) Typical horseshoe shaped ribbons are observed (open arrowheads). (E) GluK5 deficient retinae reveal to a high degree punctuated immunolabeling (arrowheads). (D’ and E’) Higher magnification of tagged frames in D (D’) and E (E’). Scale bars (A—C): 500 nm; D and E: 5 μm.</p

The absence of GluK5 disrupts the normal organization of presynaptic rod photoreceptor ribbons.

<p>The electron micrographs show sections through rod ribbon synapses in the outer plexiform layer of wild-type (A) and GluK5-knockout retinae (B-D, G). Wild-type terminals (A) show normal synaptic architecture with a synaptic ribbon anchored to the presynaptic membrane and with postsynaptic elements formed by processes of horizontal and bipolar cells. (B-D) Rod terminals in adult GluK5-/- mice showing “disintegrated” synaptic ribbons. In some cases ribbon material seems to be free-floating within the rod terminal (B and D, arrowheads). (E –F) The synaptic ribbon structure is not affected at synapses in the IPL as demonstrated for ribbons of ON-cone (E) and OFF-cone (F) bipolar cells. The lower magnification (G) shows that most of rod photoreceptor ribbons are affected in the GluK5 knockout retina. (H) Histogram showing the number of ribbon fragments at rod photoreceptor terminals in GluK5 knockout and wild type animals. n = number of presynaptic elements analysed. ***P < 0,001 Mann-Whitney-U-Test. H = horizontal cell, B = Bipolar cell, Off-CBP = off-cone bipolar cell, On-CBP = on-cone bipolar cell. All scale bars: 250 nm.</p

Immunohistochemical analysis of the mouse retina using an anti-GluK5 antibody.

<p>(A) Immunofluorescence analysis showing strong, punctate anti-GluK5 staining in the outer (arrowheads) and inner (open arrowheads) plexiform layer of the wild-type mouse retina. In the OPL (A’) the horseshoe-like appearance of the photoreceptor ribbons is clearly visible (arrowheads). (B) GluK5 immunolabeling does not colocalize with the presynaptic marker synaptophysin in the outer plexiform layer. High power photographs (B’) demonstrate adjacent localization (arrowheads). (C) Fluorescence immunostaining for CtBP2 and GluK5 in the outer plexiform layer. The horseshoe-like appearance of the photoreceptor synaptic ribbons colabels for CtBP2 and GluK5 and is clearly visible at higher magnification (arrowheads in C and C’). D) Double fluorescence immunolabeling for anti-GluK5 and anti-Calbindin, a marker for horizontal cells. The high-power photograph in D’ shows only very weak colocalization (arrowhead). (E) Double fluorescence immunolabeling with anti-PKC labeling rod bipolar cells and GluK5. GluK5 expression in the OPL does not colocalize with processes and terminals of rod bipolar cells (see higher magnification in E’). Scale bars: A—E: 10 μm; A’–E’: 2 μm.</p

Ultrastructural localization of GluK5 in the outer and inner plexiform layer of the mouse retina.

<p>(A and B) Electron micrograph of rod photoreceptor ribbons showing the ultrastructural localization of GluK5 immunoreactivity in rod spherules. Note the distribution of GluK5 immunoreactivity at the rod photoreceptor ribbon synapse covering the entire presynaptic ribbon structure (arrowheads). (C) Electron micrograph of a cone pedicle in the OPL showing GluK5 localization (arrowheads) in processes of OFF-cone bipolar cells. (D—F) Electron microscopic analysis of GluK5 distribution in the IPL. In the outer part of the inner plexiform layer GluK5 labeling is found in processes postsynaptic to OFF-cone bipolar cells (D). In the inner part of the inner plexiform layer labeling is seen in processes postsynaptic to ON-cone bipolar cells (E) and to rod bipolar cells (F). The arrows point to the presynaptic ribbon, the presynaptic terminals are surrounded with red dotted lines; the postsynaptic GluK5 positive elements are visualized with blue dotted lines. For better visualization in the electron micrographs presynaptic elements are visualized with blue and postsynaptic elements with red dotted lines. H = horizontal cell, RS = rod spherule, CP = cone pedicle, Off-CBP = off-cone bipolar cell, On-CBP = on-cone bipolar cell, RBP = rod bipolar cell. All scale bars: 100 nm.</p