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Cl(-) plays a crucial role in neuronal function and synaptic inhibition. However, the impact of neuronal morphology on the diffusion and redistribution of intracellular Cl(-) is not well understood. The role of spines in Cl(-) diffusion along dendritic trees has not been addressed so far. Because measuring fast and spatially restricted Cl(-) changes within dendrites is not yet technically possible, we used computational approaches to predict the effects of spines on Cl(-) dynamics in morphologically complex dendrites. In all morphologies tested, including dendrites imaged by super-resolution STED microscopy in live brain tissue, spines slowed down longitudinal Cl(-) diffusion along dendrites. This effect was robust and could be observed in both deterministic as well as stochastic simulations. Cl(-) extrusion altered Cl(-) diffusion to a much lesser extent than the presence of spines. The spine-dependent slowing of Cl(-) diffusion affected the amount and spatial spread of changes in the GABA reversal potential thereby altering homosynaptic as well as heterosynaptic short-term ionic plasticity at GABAergic synapses in dendrites. Altogether, our results suggest a fundamental role of dendritic spines in shaping Cl(-) diffusion, which could be of relevance in the context of pathological conditions where spine densities and neural excitability are perturbed.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
Introduction: Neuronal death and subsequent denervation of target areas are hallmarks of many neurological disorders. Denervated neurons lose part of their dendritic tree, and are considered "atrophic", i.e. pathologically altered and damaged. The functional consequences of this phenomenon are poorly understood.
Results: Using computational modelling of 3D-reconstructed granule cells we show that denervation-induced dendritic atrophy also subserves homeostatic functions: By shortening their dendritic tree, granule cells compensate for the loss of inputs by a precise adjustment of excitability. As a consequence, surviving afferents are able to activate the cells, thereby allowing information to flow again through the denervated area. In addition, action potentials backpropagating from the soma to the synapses are enhanced specifically in reorganized portions of the dendritic arbor, resulting in their increased synaptic plasticity. These two observations generalize to any given dendritic tree undergoing structural changes.
Conclusions: Structural homeostatic plasticity, i.e. homeostatic dendritic remodeling, is operating in long-term denervated neurons to achieve functional homeostasis.
Synaptic dysfunction and synapse loss are key features of Alzheimer's pathogenesis. Previously, we showed an essential function of APP and APLP2 for synaptic plasticity, learning and memory. Here, we used organotypic hippocampal cultures to investigate the specific role(s) of APP family members and their fragments for dendritic complexity and spine formation of principal neurons within the hippocampus. Whereas CA1 neurons from APLP1-KO or APLP2-KO mice showed normal neuronal morphology and spine density, APP-KO mice revealed a highly reduced dendritic complexity in mid-apical dendrites. Despite unaltered morphology of APLP2-KO neurons, combined APP/APLP2-DKO mutants showed an additional branching defect in proximal apical dendrites, indicating redundancy and a combined function of APP and APLP2 for dendritic architecture. Remarkably, APP-KO neurons showed a pronounced decrease in spine density and reductions in the number of mushroom spines. No further decrease in spine density, however, was detectable in APP/APLP2-DKO mice. Mechanistically, using APPsalpha-KI mice lacking transmembrane APP and expressing solely the secreted APPsalpha fragment we demonstrate that APPsalpha expression alone is sufficient to prevent the defects in spine density observed in APP-KO mice. Collectively, these studies reveal a combined role of APP and APLP2 for dendritic architecture and a unique function of secreted APPs for spine density.
Poster presentation: Twenty Second Annual Computational Neuroscience Meeting: CNS*2013. Paris, France. 13-18 July 2013.
Neuronal death and subsequent denervation of target areas is a major feature of several neurological conditions such as brain trauma, ischemia or neurodegeneration. The denervation-induced axonal loss results in reorganization of the dendritic tree of denervated neurons. Dendritic reorganization of denervated neurons has been previously studied using entorhinal cortex lesion (ECL).
ECL leads to shortening and loss of dendritic segments in the denervated outer molecular layer of the dentate gyrus [1]. However, the functional importance of these long-term dendritic alterations is not yet understood and their impact on neuronal electrical properties remains unclear. Therefore, in this study we analyzed what happens to the electrotonic structure and excitability of dentate granule cells after denervation-induced alterations of their dendritic morphology, assuming all other parameters remain equal.
To perform comparative electrotonic analysis we used computer simulations in anatomically and biophysically realistic compartmental models of 3D-reconstructed healthy and denervated granule cells. Our results show that somatofugal and somatopetal voltage attenuation due to passive cable properties was strongly reduced in denervated granule cells. In line with these predictions, the attenuation of simulated backpropagating action potentials and forward propagating EPSPs was significantly reduced in dendrites of denervated neurons. In addition, simulations of somatic and dendritic frequency-current (f-I) curves revealed increased excitability in deafferentated granule cells.
Taken together, our results indicate that unless counterbalanced by a compensatory adjustment of passive and/or active membrane properties, the plastic remodeling of dendrites following lesion of entorhinal cortex inputs to granule cells will boost their electrotonic compactness and excitability.
Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient.
Objective: Using multimodal imaging, we tested the hypothesis that patients after hemispherotomy recruit non-primary motor areas and non-pyramidal descending motor fibers to restore motor function of the impaired limb. Methods: Functional and structural MRI data were acquired in a group of 25 patients who had undergone hemispherotomy and in a matched group of healthy controls. Patients’ motor impairment was measured using the Fugl-Meyer Motor Assessment. Cortical areas governing upper extremity motor-control were identified by task-based functional MRI. The resulting areas were used as nodes for functional and structural connectivity analyses. Results: In hemispherotomy patients, movement of the impaired upper extremity was associated to widespread activation of non-primary premotor areas, whereas movement of the unimpaired one and of the control group related to activations prevalently located in the primary motor cortex (all p ≤ 0.05, FWE-corrected). Non-pyramidal tracts originating in premotor/supplementary motor areas and descending through the pontine tegmentum showed relatively higher structural connectivity in patients (p < 0.001, FWE-corrected). Significant correlations between structural connectivity and motor impairment were found for non-pyramidal (p = 0.023, FWE-corrected), but not for pyramidal connections. Interpretation: A premotor/supplementary motor network and non-pyramidal fibers seem to mediate motor function in patients after hemispherotomy. In case of hemispheric lesion, the homologous regions in the contralesional hemisphere may not compensate the resulting motor deficit, but the functionally redundant premotor network.
Repetitive transcranial magnetic stimulation (rTMS) is used as a therapeutic tool in neurology and psychiatry. While repetitive magnetic stimulation (rMS) has been shown to induce plasticity of excitatory synapses, it is unclear whether rMS can also modify structural and functional properties of inhibitory inputs. Here we employed 10-Hz rMS of entorhinohippocampal slice cultures to study plasticity of inhibitory neurotransmission on CA1 pyramidal neurons. Our experiments reveal a rMS-induced reduction in GABAergic synaptic strength (2–4 h after stimulation), which is Ca2+-dependent and accompanied by the remodelling of postsynaptic gephyrin scaffolds. Furthermore, we present evidence that 10-Hz rMS predominantly acts on dendritic, but not somatic inhibition. Consistent with this finding, a reduction in clustered gephyrin is detected in CA1 stratum radiatum of rTMS-treated anaesthetized mice. These results disclose that rTMS induces coordinated Ca2+-dependent structural and functional changes of specific inhibitory postsynapses on principal neurons.
Neurogenesis of hippocampal granule cells (GCs) persists throughout mammalian life and is important for learning and memory. How newborn GCs differentiate and mature into an existing circuit during this time period is not yet fully understood. We established a method to visualize postnatally generated GCs in organotypic entorhino-hippocampal slice cultures (OTCs) using retroviral (RV) GFP-labeling and performed time-lapse imaging to study their morphological development in vitro. Using anterograde tracing we could, furthermore, demonstrate that the postnatally generated GCs in OTCs, similar to adult born GCs, grow into an existing entorhino-dentate circuitry. RV-labeled GCs were identified and individual cells were followed for up to four weeks post injection. Postnatally born GCs exhibited highly dynamic structural changes, including dendritic growth spurts but also retraction of dendrites and phases of dendritic stabilization. In contrast, older, presumably prenatally born GCs labeled with an adeno-associated virus (AAV), were far less dynamic. We propose that the high degree of structural flexibility seen in our preparations is necessary for the integration of newborn granule cells into an already existing neuronal circuit of the dentate gyrus in which they have to compete for entorhinal input with cells generated and integrated earlier.
Alterations in dendritic spine numbers are linked to deficits in learning and memory. While we previously revealed that postsynaptic plasticity-related gene 1 (PRG-1) controls lysophosphatidic acid (LPA) signaling at glutamatergic synapses via presynaptic LPA receptors, we now show that PRG-1 also affects spine density and synaptic plasticity in a cell-autonomous fashion via protein phosphatase 2A (PP2A)/β1-integrin activation. PRG-1 deficiency reduces spine numbers and β1-integrin activation, alters long-term potentiation (LTP), and impairs spatial memory. The intracellular PRG-1 C terminus interacts in an LPA-dependent fashion with PP2A, thus modulating its phosphatase activity at the postsynaptic density. This results in recruitment of adhesome components src, paxillin, and talin to lipid rafts and ultimately in activation of β1-integrins. Consistent with these findings, activation of PP2A with FTY720 rescues defects in spine density and LTP of PRG-1-deficient animals. These results disclose a mechanism by which bioactive lipid signaling via PRG-1 could affect synaptic plasticity and memory formation.
Neurons which lose part of their input respond with a compensatory increase in excitatory synaptic strength. This observation is of particular interest in the context of neurological diseases, which are accompanied by the loss of neurons and subsequent denervation of connected brain regions. However, while the cellular and molecular mechanisms of pharmacologically induced homeostatic synaptic plasticity have been identified to a certain degree, denervation-induced homeostatic synaptic plasticity remains not well understood. Here, we employed the entorhinal denervation in vitro model to study the role of tumor necrosis factor alpha (TNFα) on changes in excitatory synaptic strength of mouse dentate granule cells following partial deafferentation. Our experiments disclose that TNFα is required for the maintenance of a compensatory increase in excitatory synaptic strength at 3–4 days post lesion (dpl), but not for the induction of synaptic scaling at 1–2 dpl. Furthermore, laser capture microdissection combined with quantitative PCR demonstrates an increase in TNFα-mRNA levels in the denervated zone, which is consistent with our previous finding on a local, i.e., layer-specific increase in excitatory synaptic strength at 3–4 dpl. Immunostainings for the glial fibrillary acidic protein and TNFα suggest that astrocytes are a source of TNFα in our experimental setting. We conclude that TNFα-signaling is a major regulatory system that aims at maintaining the homeostatic synaptic response of denervated neurons.
Epilepsy is a complex neurological disorder which can severely affect neuronal function. Some patients may experience status epilepticus, a life-threatening state of ongoing seizure activity associated with postictal cognitive dysfunction. However, the molecular mechanisms by which status epilepticus influences brain function beyond seizure activity remain not well understood. Here, we addressed the question of whether pilocarpine-induced status epilepticus affects synaptopodin (SP), an actin-binding protein, which regulates the ability of neurons to express synaptic plasticity. This makes SP an interesting marker for epilepsy-associated alterations in synaptic function. Indeed, single dose intraperitoneal pilocarpine injection (250 mg/kg) in three-month-old male C57BL/6J mice leads to a rapid reduction in hippocampal SP-cluster sizes and numbers (in CA1 stratum radiatum of the dorsal hippocampus; 90 min after injection). In line with this observation (and previous work using SP-deficient mice), a defect in the ability to induce long-term potentiation (LTP) of Schaffer collateral-CA1 synapses is observed. Based on these findings we propose that status epilepticus could exert its aftereffects on cognition at least in part by perturbing SP-dependent mechanisms of synaptic plasticity.
Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL/6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNFα) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNFα, IL6, IL1β, and IFNγ and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.
The entorhino-dentate projection, i.e., the perforant pathway, terminates in a highly ordered and laminated fashion in the rodent dentate gyrus (DG): fibers arising from the medial entorhinal cortex (MEC) terminate in the middle molecular layer, whereas fibers arising from the lateral entorhinal cortex (LEC) terminate in the outer molecular layer of the DG. In rats and rabbits, a crossed entorhino-dentate projection exists, which originates from the entorhinal cortex (EC) and terminates in the contralateral DG. In contrast, in mice, such a crossed projection is reportedly absent. Using single and double mouse organotypic entorhino-hippocampal slice cultures, we studied the ipsi- and crossed entorhino-dentate projections. Viral tracing revealed that entorhino-dentate projections terminate with a high degree of lamina-specificity in single as well as in double cultures. Furthermore, in double cultures, entorhinal axons arising from one slice freely intermingled with entorhinal axons originating from the other slice. In single as well as in double cultures, entorhinal axons exhibited a correct topographical projection to the DG: medial entorhinal axons terminated in the middle and lateral entorhinal axons terminated in the outer molecular layer. Finally, entorhinal neurons were virally transduced with Channelrhodopsin2-YFP and stimulated with light, revealing functional connections between the EC and dentate granule cells. We conclude from our findings that entorhino-dentate projections form bilaterally in the mouse hippocampus in vitro and that the mouse DG provides a permissive environment for crossed entorhinal fibers.
During postnatal development hippocampal dentate granule cells (GCs) often extend dendrites from the basal pole of their cell bodies into the hilar region. These so-called hilar basal dendrites (hBD) usually regress with maturation. However, hBDs may persist in a subset of mature GCs under certain conditions (both physiological and pathological). The functional role of these hBD-GCs remains not well understood. Here, we have studied hBD-GCs in mature (≥18 days in vitro) mouse entorhino-hippocampal slice cultures under control conditions and have compared their basic functional properties (basic intrinsic and synaptic properties) and structural properties (dendritic arborisation and spine densities) to those of neighboring GCs without hBDs in the same set of cultures. Except for the presence of hBDs, we did not detect major differences between the two GC populations. Furthermore, paired recordings of neighboring GCs with and without hBDs did not reveal evidence for a heavy aberrant GC-to-GC connectivity. Taken together, our data suggest that in control cultures the presence of hBDs on GCs is neither sufficient to predict alterations in the basic functional and structural properties of these GCs nor indicative of a heavy GC-to-GC connectivity between neighboring GCs.
A hallmark of several major neurological diseases is neuronal cell death. In addition to this primary pathology, secondary injury is seen in connected brain regions in which neurons not directly affected by the disease are denervated. These transneuronal effects on the network contribute considerably to the clinical symptoms. Since denervated neurons are viable, they are attractive targets for intervention. Therefore, we studied the role of Sphingosine-1-phosphate (S1P)-receptor signaling, the target of Fingolimod (FTY720), in denervation-induced dendritic atrophy. The entorhinal denervation in vitro model was used to assess dendritic changes of denervated mouse dentate granule cells. Live-cell microscopy of GFP-expressing granule cells in organotypic entorhino-hippocampal slice cultures was employed to follow individual dendritic segments for up to 6 weeks after deafferentation. A set of slice cultures was treated with FTY720 or the S1P-receptor (S1PR) antagonist VPC23019. Lesion-induced changes in S1P (mass spectrometry) and S1PR-mRNA levels (laser microdissection and qPCR) were determined. Denervation caused profound changes in dendritic stability. Dendritic elongation and retraction events were markedly increased, resulting in a net reduction of total dendritic length (TDL) during the first 2 weeks after denervation, followed by a gradual recovery in TDL. These changes were accompanied by an increase in S1P and S1PR1- and S1PR3-mRNA levels, and were not observed in slice cultures treated with FTY720 or VPC23019. We conclude that inhibition of S1PR signaling prevents dendritic destabilization and denervation-induced dendrite loss. These results suggest a novel neuroprotective effect for pharmaceuticals targeting neural S1PR pathways.
Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.
During the last 30 years, our understanding of the development and diversification of postganglionic sympathetic neurons has dramatically increased. In parallel, the list of target structures has been critically extended from the cardiovascular system and selected glandular structures to metabolically relevant tissues such as white and brown adipose tissue, lymphoid tissues, bone, and bone marrow. A critical question now emerges for the integration of the diverse sympathetic neuron classes into neural circuits specific for these different target tissues to achieve the homeostatic regulation of the physiological ends affected.
Im Jahr 1906 beschrieb Alois Alzheimer (1864 – 1915) erstmals krankhafte Eiweißablagerungen im Gehirn einer Patientin, bei der er einige Jahre zuvor eine Demenz diagnostiziert hatte. Diese Ablagerungen machte er für den geistigen Abbau verantwortlich. Über die zugrunde liegenden biologischen Ursachen der Krankheit (»Ätiologie«) konnte der Frankfurter Arzt jedoch nur Vermutungen anstellen. Inzwischen weiß man, dass die Gene mit darüber entscheiden, ob jemand im Alter an Alzheimer-Demenz (AD) erkrankt. Bei der seltener auftretenden familiären Form der AD sind die verantwortlichen Gene inzwischen bekannt. Doch auch bei der häufigeren sporadischen Form der Krankheit konnten verschiedene Arbeitsgruppen, einschließlich unserer eigenen, inzwischen einige »Risiko-Gene« identifizieren. Eine Erkrankung des Gehirns Aufbauend auf den Befunden von Alois Alzheimer beschäftigten sich in der zweiten Hälfte des 20. Jahrhunderts immer mehr Forschergruppen mit der Alzheimer-Krankheit. ...
Large spines are stable and important for memory trace formation. The majority of large spines also contains synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using 2-photon time-lapse imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive (SP+) spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP+ spines followed conditional two-stage decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single-phase exponential decays. This was also the case following afferent denervation. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines, and the presence of SP indicates spines of high stability.