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The hyperexcitability of dentate granule neurons in organotypic hippocampal slice cultures is due to reorganization of synaptic inputs in vitro.

ABSTRACT: Organotypic hippocampal slice cultures (OHSCs) provide the experimental flexibility of cell culture while leaving much of the natural neuronal connectivity intact. Previously, it was shown that the functional and morphological features of CA1 pyramidal neurons in OHSCs resemble, to a surprising extent, those of CA1 neurons in the acute brain slice preparation. However, the extent to which the characteristics of other principle hippocampal neurons change or are preserved in cultured slices remains to be determined. In the present study, I initially sought to understand whether and how the synaptic inputs and morphology of cultured dentate granule neurons (GCs) differ from GCs that have developed in vivo. To this end, I compared GCs in OHSCs and GCs in acute slices at two equivalent developmental time points (P14 vs. DIV7 and P21 vs. DIV21). The findings suggest that there is considerable reorganization of synaptic input to the organotypic GCs, such that these cells are more susceptible to hyperexcitation than GCs in acute slices after 3 weeks. It appears that this hyperexcitability emerges through an increase in the proportion of mature synapses at proximal dendritic sites and is accompanied by an increase in inhibitory neuron activity. These alterations appear to arise in a coordinated manner such that the substantial increase in excitatory synaptic drive received by the DIV21 GCs in OHSCs remains local and is not translated into excessive output possibly leading to damage or major morphological alterations of downstream pyramidal neurons.

SUBMITTER: Gilbride CJ

PROVIDER: S-EPMC5064129 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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The hyperexcitability of dentate granule neurons in organotypic hippocampal slice cultures is due to reorganization of synaptic inputs in vitro.

Gilbride Charlie J CJ

Physiological reports 20161001 19

Organotypic hippocampal slice cultures (OHSCs) provide the experimental flexibility of cell culture while leaving much of the natural neuronal connectivity intact. Previously, it was shown that the functional and morphological features of CA1 pyramidal neurons in OHSCs resemble, to a surprising extent, those of CA1 neurons in the acute brain slice preparation. However, the extent to which the characteristics of other principle hippocampal neurons change or are preserved in cultured slices remain ...[more]

PMID: 27707779

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Project description:Neurogenesis in the adult hippocampus has become an intensively investigated research topic, as it is essential for proper hippocampal function and considered to bear therapeutic potential for the replacement of pathologically lost neurons. On the other hand, neurogenesis itself is frequently affected by CNS insults. To identify processes leading to the disturbance of neurogenesis, we made use of organotypic hippocampal slice cultures (OHSC), which, for unknown reasons, lose their neurogenic potential during cultivation. In the present study, we show by BrdU/Prox1 double-immunostaining that the generation of new granule cells drops by 90% during the first week of cultivation. Monitoring neurogenesis dynamically in OHSC from POMC-eGFP mice, in which immature granule cells are endogenously labeled, revealed a gradual decay of the eGFP signal, reaching 10% of initial values within 7 days of cultivation. Accordingly, reverse transcription quantitative polymerase chain reaction analysis showed the downregulation of the neurogenesis-related genes doublecortin and Hes5, a crucial target of the stem cell-maintaining Notch signaling pathway. In parallel, we demonstrate a strong and long-lasting activation of astrocytes and microglial cells, both, morphologically and on the level of gene expression. Enhancement of astroglial activation by treating OHSC with ciliary neurotrophic factor accelerated the loss of neurogenesis, whereas treatment with indomethacin or an antagonist of the purinergic P2Y12 receptor exhibited potent protective effects on the neurogenic outcome. Therefore, we conclude that OHSC rapidly lose their neurogenic capacity due to persistent inflammatory processes taking place after the slice preparation. As inflammation is also considered to affect neurogenesis in many CNS pathologies, OHSC appear as a useful tool to study this interplay and its molecular basis. Furthermore, we propose that modification of glial activation might bear the therapeutic potential of enabling neurogenesis under neuropathological conditions.

Project description:Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

Project description:BACKGROUND:Pyrethroids, such as bifenthrin (BF), are among the most widely used class of insecticides that pose serious risks to human and wildlife health. Pyrethroids are proposed to affect astrocytic functions and to cause neuron injury in the central nervous system (CNS). Microglia are key cells involved in innate immune responses in the CNS, and microglia activation has been linked to inflammation and neurotoxicity. However, little information is known about the effects of BF-induced toxicity in primary microglial cells as well as in organotypic hippocampal slice cultures (OHSCs). METHODS:Oxidative stress and inflammatory responses induced by BF were evaluated in primary microglial cells and OHSCs incubated with different concentrations of BF (1-20 ?M) for 4 and 24 h. mRNA and protein synthesis of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), nuclear erythroid-2 like factor-2 (Nrf-2), and microsomal prostaglandin synthase-1 (mPGES-1) was also studied by qPCR and Western blot. Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Neurotoxicity in OHSCs was analyzed by propidium iodide (PI) staining and confocal microscopy. RESULTS:Exposure of microglial cells to BF for 24 h resulted in a dose-dependent reduction in the number of viable cells. At sub-cytotoxic concentrations, BF increased reactive oxygen species (ROS), TNF-alpha synthesis, and prostaglandin E2 (PGE2) production, at both 4- and 24-h time points, respectively. Furthermore, BF incubation decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and increased lipid peroxidation, protein oxidation, and H2O2 formation. In addition, BF significantly induced protein synthesis and mRNA expression of oxidative and inflammatory mediators after 4 and 24 h, including Nrf-2, COX-2, mPGES-1, and nuclear factor kappaB (NF-kappaB). A 24-h exposure of OHSCs to BF also increased neuronal death compared to untreated controls. Furthermore, depletion of microglia from OHSCs potently enhanced neuronal death induced by BF. CONCLUSIONS:Overall, BF exhibited cytotoxic effects in primary microglial cells, accompanied by the induction of various inflammatory and oxidative stress markers including the Nrf-2/COX-2/mPGES-1/NF-kappaB pathways. Moreover, the study provided evidence that BF induced neuronal death in OHSCs and suggests that microglia exert a protective function against BF toxicity.

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The hyperexcitability of dentate granule neurons in organotypic hippocampal slice cultures is due to reorganization of synaptic inputs in vitro.

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The hyperexcitability of dentate granule neurons in organotypic hippocampal slice cultures is due to reorganization of synaptic inputs in vitro.

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