Bernd Heimrich

Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors.

Glutamate receptors of the AMPA-subtype (AMPARs), together with the transmembrane AMPAR regulatory proteins (TARPs), mediate fast excitatory synaptic transmission in the mammalian brain. Here, we show by proteomic analysis that the majority of AMPARs in the rat brain are coassembled with two members of the cornichon family of transmembrane proteins, rather than with the TARPs. Coassembly with cornichon homologs 2 and 3 affects AMPARs in two ways: Cornichons increase surface expression of AMPARs, and they alter channel gating by markedly slowing deactivation and desensitization kinetics. These results demonstrate that cornichons are intrinsic auxiliary subunits of native AMPARs and provide previously unknown molecular determinants for glutamatergic neurotransmission in the central nervous system.

Sprouting in the hippocampus is layer-specific.

Partial removal of layer-specific afferents of the hippocampus is said to induce sprouting of intact fibers from neighboring layers that invade the zone of the degenerating axons. However, recent in vivo and in vitro studies using sensitive anterograde tracers have failed to demonstrate sprouting across laminar boundaries. Sprouting does occur; but, it mainly involves unlesioned fiber systems terminating within the layer of fiber degeneration in addition to the degenerating afferents. These findings point to rigid laminar cues attracting certain fiber systems while repelling others in normal development and after partial deafferentation.

The Repulsive Guidance Molecule RGMa Is Involved in the Formation of Afferent Connections in the Dentate Gyrus

In the developing dentate gyrus, afferent fiber projections terminate in distinct laminas. This relies on an accurately regulated spatiotemporal network of guidance molecules. Here, we have analyzed the functional role of the glycosylphosphatidylinositol (GPI)-anchored repulsive guidance molecule RGMa. In situ hybridization in embryonic and postnatal brain showed expression of RGMa in the cornu ammonis and hilus of the hippocampus. In the dentate gyrus, RGM immunostaining was confined to the inner molecular layer, whereas the outer molecular layers targeted by entorhinal fibers remained free. To test the repulsive capacity of RGMa, different setups were used: the stripe and explant outgrowth assays with recombinant RGMa, and entorhino–hippocampal cocultures incubated either with a neutralizing RGMa antibody (Ab) or with the GPI anchor-digesting drug phosphatidylinositol-specific phospholipase C. Entorhinal axons were clearly repelled by RGMa in the stripe and outgrowth assays. After disrupting the RGMa function, the specific laminar termination pattern in entorhino–hippocampal cocultures was lost, and entorhinal axons entered inappropriate hippocampal areas. Our data indicate an important role of RGMa for the layer-specific termination of the perforant pathway as a repulsive signal that compels entorhinal fibers to stay in their correct target zone.

Molecular analysis of Nogo expression in the hippocampus during development and following lesion and seizure

The Nogo gene encodes an integral membrane protein mainly responsible for the neurite inhibition properties of myelin. Here, we analyzed the expression pattern of Nogo-A, Nogo-B, and Nogo-C and Nogo-66 receptor (Ng66R) mRNA during hippocampal development and lesion-induced axonal sprouting. Nogo-A and Nogo-B and Ng66R transcripts preceded the progress of myelination and were highly expressed at postnatal day zero (P0) in all principal hippocampal cell layers, with the exception of dentate granule cells. Only a slight Nogo-C expression was found at P0 in the principal cell layers of the hippocampus. During adulthood, all Nogo splice variants and their receptor were expressed in the neuronal cell layers of the hippocampus, in contrast to the myelin basic protein mRNA expression pattern, which revealed a neuronal source of Nogo gene expression in addition to oligodendrocytes. After hippocampal denervation, the Nogo genes showed an isoform-specific temporal regulation. All Nogo genes were strongly regulated in the hippocampal cell layers, whereas the Ng66R transcripts showed a significant increase in the contralateral cortex. These data could be confirmed on protein levels. Furthermore, Nogo-A expression was up-regulated after kainate-induced seizures. Our data show that neurons express Nogo genes with a clearly distinguishable pattern during development. This expression is further dynamically and isoform-specifically altered after lesioning during the early phase of structural rearrengements. Thus, our results indicate a role for Nogo-A, -B, and -C during development and during the stabilization phase of hippocampal reorganization. Taken together with these data, the finding that neurons in a highly plastic brain region express Nogo genes supports the hypothesis that Nogo may function beyond its known neuronal growth inhibition activity in shaping neuronal circuits.

Developmental distribution of a reeler gene-related antigen in the rat hippocampal formation visualized by CR-50 immunocytochemistry.

During histogenesis of the neocortex, Cajal Retzius cells in the marginal zone express the glycoprotein reelin which is developmentally regulated and involved in the formation of the inside out mode of cortical layering. Cajal Retzius cells are also present in the developing hippocampus. There, inhibition of reelin by blocking with CR-50, an antibody which recognizes the N-terminus of this protein, leads to abnormal development of layer-specific connections. Here we report the developmental distribution pattern of reelin expressing neurons in the rat hippocampal formation using CR-50 immunocytochemistry. Labelled Cajal Retzius cells were located near the hippocampal fissure in neonate rats. Many of these cells were still present in the adult. From postnatal day 4 on, neurons in other layers were stained with the CR-50 antibody. In adult rats immunopositive neurons were found in all hippocampal subfields and in the entorhinal cortex. These observations indicate that in the rat hippocampal formation reelin is expressed in different neuronal types during development and in adulthood. Moreover, Cajal Retzius cells in the marginal zone near the hippocampal fissure are still found in adult animals.

Plasticity of identified neurons in slice cultures of hippocampus : a combined Golgi/electron microscopic and immunocytochemical study

The combined Golgi/electron microscope (EM) technique and immunocytochemistry for glutamate decarboxylase (GAD) were used to study the differentiation of pyramidal neurons and GABAergic inhibitory non-pyramidal cells in slice cultures of rat and mouse hippocampus. Golgi-impregnated and gold-toned cultures showed the characteristic curved structure of the Ammons horn. Hippocampal regions CA1, CA3 and fascia dentata could easily be recognized. Pyramidal neurons in CA1 displayed all characteristics of this cell type known from Golgi studies in situ. A triangular cell body gives rise to a main apical dendritic shaft which gives off several side branches. Basal dendrites and the axon originate at the basal pole of the cell body. Apical and basal dendrites are densely covered with spines. As a characteristic feature of the cultured pyramidal cells, numerous spines were observed on the cell body. Most likely due to flattening of the slice during incubation, the pyramidal neurons in CA1 are no longer arranged in a densely packed layer. This results in more space between cell bodies which is filled in by numerous horizontal and basal dendrites originating from the pyramidal cell perikaryon. CA1 pyramidal neurons in slice cultures of the rat or mouse thus resemble the pyramidal neurons in the CA1 region of the primate hippocampus where a similar loose distribution of cell bodies is found. In the electron microscope, cell bodies and dendritic shafts of the gold-toned pyramidal cells formed symmetric synaptic contacts with presynaptic terminals. Numerous boutons were observed that established asymmetric synaptic contacts on gold-toned spines of peripheral pyramidal cell dendrites. This suggests that considerable synaptic reorganization takes place because in situ spines on peripheral dendritic segments are contacted mainly by extrinsic afferents. Like in situ, at least some of the terminals that establish symmetric synaptic contacts are GABAergic. In our immunocytochemical study we observed numerous GAD-positive terminals that formed a dense pericellular plexus around immunonegative cell bodies of pyramidal neurons. In the electron microscope these structures were identified as presynaptic boutons which formed symmetric synaptic contacts on cell bodies and dendritic shafts. They most likely originated from the GAD-positive neurons scattered in all layers of the slice culture. Our results have shown that the main cell types in the hippocampus, pyramidal neurons and GABAergic inhibitory non-pyramidal cells, survive and differentiate under the present culture conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies.

Cajal–Retzius (CR) cells are characteristic horizontally orientated, early‐generated transient neurons in the marginal zones of the neocortex and hippocampus that synthesize the extracellular matrix protein reelin. They have been implicated in the pathfinding of entorhino‐hippocampal axons, but their role in this process remained unclear. Here we have studied the axonal projection of hippocampal CR cells. Following injection of the carbocyanine dye DiI into the entorhinal cortex of aldehyde‐fixed rat embryos and young postnatal rats, neurons in the outer molecular layer of the dentate gyrus and stratum lacunosum‐moleculare of the hippocampus proper with morphological characteristics of CR cells were retrogradely labelled. In a time course analysis, the first retrogradely labelled CR cells were observed on embryonic day 17. This projection of hippocampal CR cells to the entorhinal cortex was confirmed by retrograde tracing with Fast Blue in new‐born rats and by intracellular biocytin filling of CR cells in acute slices from young postnatal rat hippocampus/entorhinal cortex and in entorhino‐hippocampal slice cocultures using infrared videomicroscopy in combination with the patch‐clamp technique. In double‐labelling experiments CR cells were identified by their immunocytochemical staining for reelin or calretinin, and their interaction with entorhino‐hippocampal axons labelled by anterograde tracers was analysed. Future studies need to investigate whether this early transient projection of hippocampal CR cells to the entorhinal cortex is used as a template by the entorhinal axons growing to their target layers in the hippocampus.

Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity

The cytopathogenicity of vesicular stomatitis virus (VSV) has been attributed mainly to the host shut-off activity of the viral matrix (M) protein, which inhibits both nuclear transcription and nucleocytoplasmic RNA transport, thereby effectively suppressing the synthesis of type I interferon (IFN). The M protein from persistently VSV-infected cells was shown to harbour characteristic amino acid substitutions (M51R, V221F and S226R) implicated in IFN induction. This study demonstrates that infection of human fibroblasts with recombinant VSV containing the M51R substitution resulted in IFN induction, whereas neither the V221F nor the S226R substitution effected an IFN-inducing phenotype. Only when V221F was combined with S226R were the host shut-off activity of the M protein abolished and IFN induced, independently of M51R. The M33A substitution, previously implicated in VSV cytotoxicity, did not affect host shut-off activity. M-mutant VSV containing all four amino acid substitutions retained cytotoxic properties in both Vero cells and IFN-competent primary fibroblasts. Infected-cell death was associated with the formation of giant polynucleated cells, suggesting that the fusion activity of the VSV G protein was involved. Accordingly, M-mutant VSV expressing a fusion-defective G protein or with a deletion of the G gene showed significantly reduced cytotoxic properties and caused long-lasting infections in Vero cells and mouse hippocampal slice cultures. In contrast, a G-deleted VSV expressing wild-type M protein remained cytotoxic. These findings indicate that the host shut-off activity of the M protein dominates VSV cytotoxicty, whilst the fusion-active G protein is mainly responsible for the cytotoxicity remaining with M-mutant VSV.

Blockade of neuronal activity alters spine maturation of dentate granule cells but not their dendritic arborization

Organotypic co-cultures of the entorhinal cortex and hippocampus were examined to determine the role of the entorhinal fibers in the dendritic development and formation of spines of dentate granule cells. Quantitative analysis of Golgi-impregnated granule cells in single hippocampal cultures and co-cultures with the entorhinal cortex revealed that the presence of entorhinal fibers promoted the elongation and differentiation of the target granule cell dendrites. This was accompanied by an increase in the total number of spines. The contribution of neuronal activity to this afferent-mediated dendritic development was tested by chronic application of the sodium channel blocker tetrodotoxin for 20 days in vitro. Tracing with biocytin showed that the formation of the entorhinohippocampal pathway was unaffected by the blockade of neuronal activity. The dendritic arbor of cultured granule cells and the number of dendritic spines did not differ between tetrodotoxin-treated slices and untreated controls. However, there was a significant increase in the relative number of filiform spines on granule cell dendrites in tetrodotoxin-treated co-cultures. Such filiform spines are a characteristic feature of immature neurons. These results suggest the cooperation of two mechanisms in the dendritic development of dentate granule cells: the specific afferent-mediated dendritic arborization and the activity-dependent maturation of spines.

The hippocampus of the reeler mutant mouse : Fiber segregation in area CA1 depends on the position of the postsynaptic target cells

Area CA1 of the rodent hippocampus is characterized by a highly lamina-specific and nonoverlapping termination of afferent fiber tracts. Entorhinal fibers terminate in stratum lacunosum-moleculare and commissural/associational fibers terminate in strata radiatum and oriens. It has been hypothesized that this fiber lamination depends on specific signals for the different afferent fiber tracts that are located on distinct dendritic segments of the postsynaptic neuron. In order to test this hypothesis, entorhinal and commissural/associational afferents to Ammons horn were investigated in the adult reeler mutant mouse, in which developmental cell migration defects have disrupted the normal array of cells. Golgi staining revealed a deep and a superficial principal cell layer in the mutant. The morphology of the cells located in the deep pyramidal cell layer was considerably abnormal, whereas most cells located in the superficial pyramidal cell layer showed an almost normal cellular and dendritic morphology. Anterograde tracing with Phaseolus vulgaris leukoagglutinin revealed that the duplication and disorganization of the pyramidal cell layer in area CA1 are mirrored by the duplication and disruption of afferent fiber termination zones. In the zone above the abnormal deep pyramidal cell layer, i.e., between the two cell layers, entorhinal fibers as well as commissural/associational fibers terminate and intermingle. In contrast, in the zone above the fairly normal superficial pyramidal cell layer, entorhinal and commissural/associational fibers retain their normal fiber segregation. These data suggest that the normal laminar organization of the murine hippocampus depends on positional cues presented by their target cells.