Tetrodotoxin prevents heat-shock induced granule cell dispersion in hippocampal slice cultures.

Granule cell dispersion (GCD) has been associated as a pathological feature of temporal lobe epilepsy (TLE). Early-life epileptiform activity such as febrile seizures has been proposed to have a causal link to developing chronic TLE. During postnatal development, the hippocampus may be particularly vulnerable to hyperexcitability-induced insults since neuronal migration and differentiation are still ongoing in the hippocampus. Further, the extracellular matrix (ECM), here in particular the protein reelin, has been implicated in the pathophysiology of GCD. Thus, loss of reelin-expressing cells, Cajal-Retzius cells and subsets of interneurons, may be related to GCD. To study the possible role of febrile seizures, we previously induced GCD in vitro by subjecting hippocampal slice cultures to a transient heat-shock, which was not accompanied by loss of Cajal-Retzius cells. In order to examine the mechanisms involved in heat-shock induced GCD, the present study aimed to determine whether such dispersion could be prevented by blocking cellular electrical activity. Here we show that the extent of heat-shock induced GCD could be significantly reduced by treatment with the sodium channel blocker tetrodotoxin (TTX), suggesting that electrical activity is an important factor involved in heat-shock induced GCD.

Heat-Shock Induces Granule Cell Dispersion and Microgliosis in Hippocampal Slice Cultures.

Granule cell dispersion (GCD) has been found in the dentate gyrus (dg) of patients with temporal lobe epilepsy (TLE) and a history of febrile seizures but was also recently observed in pediatric patients that did not suffer from epilepsy. This indicates that GCD might not always be disease related, but instead could reflect normal morphological variation. Thus, distribution of newborn granule cells within the hilar region is part of normal dg development at early stages but could be misinterpreted as pathological GCD. In turn, pathological GCD may be caused, for example, by genetic mutations, such as the reeler mutation. GCD in the reeler mutant goes along with an increased susceptibility to epileptiform activity. Pathological GCD in combination with epilepsy is caused by experimental administration of the glutamate receptor agonist kainic acid in rodents. In consequence, the interpretation of GCD and the role of febrile seizures remain controversial. Here, we asked whether febrile temperatures alone might be sufficient to trigger GCD and used hippocampal slice cultures as in vitro model to analyze the effect of a transient temperature increase on the dg morphology. We found that a heat-shock of 41°C for 6 h was sufficient to induce GCD and degeneration of a fraction of granule cells. Both of these factors, broadening of the granule cell layer (gcl) and increased neuronal cell death within the gcl, contributed to the development of a significantly reduced packaging density of granule cells. In contrast, Reelin expressing Cajal-Retzius (CR) cells in the molecular layer were heat-shock resistant. Thus, their number was not reduced, and we did not detect degenerating CR cells after heat-shock, implying that GCD was not caused by the loss of CR cells. Importantly, the heat-shock-induced deterioration of dg morphology was accompanied by a massive microgliosis, reflecting a robust heat-shock-induced immune response. In contrast, in the study that reported on GCD as a non-specific finding in pediatric patients, no microglia reaction was observed. Thus, our findings underpin the importance of microglia as a marker to distinguish pathological GCD from normal morphological variation.

Reelin signaling modulates GABAB receptor function in the neocortex.

Reelin is a protein that is best known for its role in controlling neuronal layer formation in the developing cortex. Here, we studied its role for post-natal cortical network function, which is poorly explored. To preclude early cortical migration defects caused by Reelin deficiency, we used a conditional Reelin knock-out (RelncKO ) mouse, and induced Reelin deficiency post-natally. Induced Reelin deficiency caused hyperexcitability of the neocortical network in vitro and ex vivo. Blocking Reelin binding to its receptors ApoER2 and VLDLR resulted in a similar effect. Hyperexcitability in RelncKO organotypic slice cultures could be rescued by co-culture with wild-type organotypic slice cultures. Moreover, the GABAB receptor (GABAB R) agonist baclofen failed to activate and the antagonist CGP35348 failed to block GABAB Rs in RelncKO mice. Immunolabeling of RelncKO cortical slices revealed a reduction in GABAB R1 and GABAB R2 surface expression at the plasma membrane and western blot of RelncKO cortical tissue revealed decreased phosphorylation of the GABAB R2 subunit at serine 892 and increased phosphorylation at serine 783, reflecting receptor deactivation and proteolysis. These data show a role of Reelin in controlling early network activity, by modulating GABAB R function. Cover Image for this issue: https://doi.org/10.1111/jnc.15054.

Distal Dendritic Enrichment of HCN1 Channels in Hippocampal CA1 Is Promoted by Estrogen, but Does Not Require Reelin.

HCN1 compartmentalization in CA1 pyramidal cells, essential for hippocampal information processing, is believed to be controlled by the extracellular matrix protein Reelin. Expression of Reelin, in turn, is stimulated by 17ß-estradiol (E2). In this study, we therefore tested whether E2 regulates the compartmentalization of HCN1 in CA1 via Reelin. In organotypic entorhino-hippocampal cultures, we found that E2 promotes HCN1 distal dendritic enrichment via the G protein-coupled estrogen receptor GPER1, but apparently independent of Reelin, because GST-RAP, known to reduce Reelin signaling, did not prevent E2-induced HCN1 enrichment in distal CA1. We therefore re-examined the role of Reelin for the regulation of HCN1 compartmentalization and could not detect effects of reduced Reelin signaling on HCN1 distribution in CA1, either in the (developmental) slice culture model or in tamoxifen-inducible conditional reelin knockout mice during adulthood. We conclude that for HCN1 channel compartmentalization in CA1 pyramidal cells, Reelin is not as essential as previously proposed, and E2 effects on HCN1 distribution in CA1 are mediated by mechanisms that do not involve Reelin. Because HCN1 localization was not altered at different phases of the estrous cycle, gonadally derived estradiol is unlikely to regulate HCN1 channel compartmentalization, while the pattern of immunoreactivity of aromatase, the final enzyme of estradiol synthesis, argues for a role of local hippocampal E2 synthesis.

Reelin and aromatase cooperate in ovarian follicle development.

Reelin plays an important role in cerebral cortex development and synaptogenesis. In the hippocampus, the neurosteroid estrogen affects reelin expression. In this study we tested a potential crosstalk between estradiol and reelin, thus the possibility of a reelin-induced activation of the estradiol synthesizing enzyme aromatase. As a model system, we used ovaries, which express reelin and are a major source of estradiol. We found that in wild-type mice, reelin and aromatase are expressed in granulosa cells of growing follicles. The expression of reelin varies with the estrus cycle and is highest shortly before ovulation, when estradiol serum levels are at their maximum. In ovaries of reelin-deficient reeler mice, aromatase mRNA and protein are significantly reduced, as evidenced by real-time PCR, western blot analysis, and quantitative immunohistochemistry in granulosa cells of preovulatory follicles. In line with reduced estradiol synthesis, ovarian estrus cycle length is prolonged in reeler mice. Most importantly, treating cultured granulosa cells with recombinant reelin results in significant upregulation of aromatase mRNA and protein and increased secretion of estradiol into the supernatant. Our data provide evidence of a local increase of aromatase expression by reelin. Regarding reproduction, this crosstalk may contribute to follicular stability and counteract luteinization in ovaries.

A 3D-matrigel/microbead assay for the visualization of mechanical tractive forces at the neurite-substrate interface of cultured neurons.

Mechanical properties of neuronal processes contribute to neuronal function, to resistance of fiber tracts against mechanical trauma, and to morphological changes during development and neurodegeneration. Conventional in vitro cell culture systems on inflexible substrates do not allow for the visualization of changing mechanical stress between neurites and their substrate. To solve this problem, we adapted a three-dimensional gel matrix assay to visualize mechanical traction forces at the neurite-substrate interface. We chose matrigel as substrate because in this matrix various types of neurons initially adapt a bipolar morphology while migrating, similar to migrating neurons in vivo. To visualize emerging traction forces between neurites and their substrate, microbeads were embedded into the matrix as visible landmarks. We first analyzed mechanical distortion of matrigel by stepwise movements of a glass pipette tip under control of a micromanipulator to ensure reproducibility of induced bead displacement. The assay was then used to study the effect of the microtubule disrupting drug nocodazole on neuronal processes. By monitoring displacement of matrigel-embedded microbeads, we visualized here for the first time emerging mechanical traction forces between the leading process and the substrate during nocodazole-induced soma translocation. We did not observe bead displacement by processes of aged neurons.

Reelin and the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promote dendritic Golgi translocation in hippocampal neurons.

In the cerebral cortex of reeler mutant mice lacking reelin expression, neurons are malpositioned and display misoriented apical dendrites. Neuronal migration defects in reeler have been studied in great detail, but how misorientation of apical dendrites is related to reelin deficiency is poorly understood. In wild-type mice, the Golgi apparatus transiently translocates into the developing apical dendrite of radially migrating neurons. This dendritic Golgi translocation has recently been shown to be promoted by reelin. However, the underlying signalling mechanisms are largely unknown. Here, we show that the Cdc42/Rac1 guanine nucleotide exchange factor αPIX/Arhgef6 promoted translocation of Golgi cisternae into developing dendrites of hippocampal neurons. Reelin treatment further increased the αPIX-dependent effect. In turn, overexpression of exchange activity-deficient αPIX or dominant-negative (dn) Cdc42 or dn-Rac1 impaired dendritic Golgi positioning, an effect that was not compensated by reelin treatment. Together, these data suggest that αPIX may promote dendritic Golgi translocation, as a downstream component of a reelin-modulated signalling pathway. Finally, we found that reelin promoted the translocation of the Golgi apparatus into the dendrite that was most proximal to the reelin source. The distribution of reelin may thus contribute to the selection of the process that becomes the apical dendrite.

Reelin promotes microtubule dynamics in processes of developing neurons.

The extracellular matrix protein reelin controls radial migration and layer formation of cortical neurons, in part by modulation of cytoskeletal dynamics. A stabilizing effect of reelin on the actin cytoskeleton has been described recently. However, it is poorly understood how reelin modulates microtubule dynamics. Here, we provide evidence that reelin increases microtubule assembly. This effect is mediated, at least in part, by promoting microtubule plus end dynamics in processes of developing neurons. Thus, we treated primary neuronal cultures with nocodazole to disrupt microtubules. After nocodazole washout, we found microtubule reassembly to be accelerated in the presence of reelin. Moreover, we show that reelin treatment promoted the formation of microtubule plus end binding protein 3 (EB3) comets in developing dendrites, and that EB3 immunostaining in the developing wild-type neocortex is most intense in the reelin-rich marginal zone where leading processes of radially migrating neurons project to. This characteristic EB3 staining pattern was absent in reeler. Also reassembly of nocodazole-dispersed dendritic Golgi apparati, which are closely associated to microtubules, was accelerated by reelin treatment, though with a substantially slower time course when compared to microtubule reassembly. In support of our in vitro results, we found that the subcellular distribution of α-tubulin and acetylated tubulin in reeler cortical sections differed from wild-type and from mice lacking the very low density lipoprotein receptor (VLDLR), known to bind reelin. Taken together, our results suggest that reelin promotes microtubule assembly, at least in part, by increasing microtubule plus end dynamics.

Congruence of vascular network remodeling and neuronal dispersion in the hippocampus of reelin-deficient mice.

In the hippocampus, neurons and fiber projections are strictly organized in layers and supplied with oxygen via a vascular network that also develops layer-specific characteristics in wild-type mice, as shown in the present study for the first time in a quantitative manner. By contrast, in the reeler mutant, well known for its neuronal migration defects due to the lack of the extracellular matrix protein reelin, emerging layer-specific characteristics of the vascular pattern were found to be remodeled during development of the dentate gyrus. Remarkably, in the first postnatal week, when a granule cell layer was still discernable in the reeler dentate gyrus, also the reeler vascular pattern resembled wild type. Thus, at postnatal day 6, unbranched microvessels traversed the granule cell layer and bifurcated when reaching the subgranular zone. Only after the first postnatal week vascular network remodeling in the reeler dentate gyrus became apparent, when the proportion of dispersed granule cells increased. Hence, vessel bifurcation frequency decreased in the maturing reeler dentate gyrus, but increased in wild type, resulting in significant differences (approx. 100%; p < 0.01) between adult wild type and reeler. Moreover, layer-specific vessel bifurcation frequencies disappeared in the maturing reeler dentate gyrus. Finally, a wild type-like vascular pattern was also found in the dentate gyrus of mice deficient for the reelin receptor very low density lipoprotein receptor (VLDLR), precluding a requirement of VLDLR for normal vascular pattern formation in the dentate gyrus. In sum, our findings show that vascular network remodeling in the reeler dentate gyrus is closely linked to the progression of granule cell dispersion.

Developmental changes in dendritic shape and synapse location tune single-neuron computations to changing behavioral functions.

During nervous system development, different classes of neurons obtain different dendritic architectures, each of which receives a large number of input synapses. However, it is not clear whether synaptic inputs are targeted to specific regions within a dendritic tree and whether dendritic tree geometry and subdendritic synapse distributions might be optimized to support proper neuronal input-output computations. This study uses an insect model where structure and function of an individually identifiable neuron, motoneuron 5 (MN5), are changed while it develops from a slow larval crawling into a fast adult flight motoneuron during metamorphosis. This allows for relating postembryonic dendritic remodeling of an individual motoneuron to developmental changes in behavioral function. Dendritic architecture of MN5 is analyzed by three-dimensional geometric reconstructions and quantitative co-localization analysis to address the distribution of synaptic terminals. Postembryonic development of MN5 comprises distinct changes in dendritic shape and in the subdendritic distribution of GABAergic input synapses onto MN5. Subdendritic synapse targeting is not a consequence of neuropil structure but must rely on specific subdendritic recognition mechanisms. Passive multicompartment simulations indicate that postembryonic changes in dendritic architecture and in subdendritic input synapse distributions may tune the passive computational properties of MN5 toward stage-specific behavioral requirements.

PTX-induced hyperexcitability affects dendritic shape and GABAergic synapse density but not synapse distribution during Manduca postembryonic motoneuron development.

During the metamorphosis of the holometabolous insect, Manduca sexta, the postembryonic acquisition of adult specific motor behaviors is accompanied by changes in dendritic architecture, membrane currents, and input synapses of identified motoneurons. This study aims to test whether increased activity affects dendritic architecture and sub-dendritic input synapse distribution of the identified flight motoneuron 5 (MN5). Systemic injections of the chloride channel blocker, picrotoxin (PTX), during early pupal stages increase pupal reflex responsiveness, but overall development is not impaired. MN5 input resistance, resting membrane potential, and spiking threshold are not affected. Bath application of PTX to isolated ventral nerve cords evokes spiking in pupal and adult flight motoneurons. Quantitative three-dimensional reconstructions of the dendritic tree of the adult MN5 show that systemic PTX injections into early pupae cause dendritic overgrowth and reduce the density of GABAergic inputs. In contrast, the distribution patterns of GABAergic terminals throughout the dendritic tree remain unaltered. This indicates that increased overall excitability might cause dendritic overgrowth and decreased inhibitory input during postembryonic motoneuron remodeling, whereas sub-dendritic synapse targeting might be controlled by activity-independent signals. Behavioral testing reveals that these neuronal changes do not impede the animal's ability to fly, but impair maximum flight performance.

Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor.

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin-related peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161-172), including the original [Asn(13)]-orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da). The fifth peptide is a hitherto unknown orcokinin variant: [Ala(8)-Ala(13)]-orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well-studied system.