In vitro ictogenesis is stochastic at the single neuron level

Seizure initiation is the least understood and most disabling element of epilepsy. Studies of ictogenesis require high speed recordings at cellular resolution in the area of seizure onset. However, in vivo seizure onset areas can’t be determined at the level of resolution necessary to enable such studies. To circumvent these challenges, we used novel GCaMP7-based calcium imaging in the organotypic hippocampal slice culture model of post-traumatic epilepsy in mice. Organotypic hippocampal slice cultures generate spontaneous, recurrent seizures in a preparation in which it is feasible to image the activity of the entire network (with no unseen inputs existing). Chronic calcium imaging of the entire hippocampal network, with paired electrophysiology, revealed 3 patterns of seizure onset: low amplitude fast activity, sentinel spike, and spike burst + low amplitude fast activity onset. These patterns recapitulate common features of human seizure onset, including low voltage fast activity and spike discharges. Weeks-long imaging of seizure activity showed a characteristic evolution in onset type and a refinement of the seizure onset zone. Longitudinal tracking of individual neurons revealed that seizure onset is stochastic at the single neuron level, suggesting that seizure initiation activates neurons in non-stereotyped sequences seizure to seizure. This study demonstrates for the first time that transitions to seizure are not initiated by a small number of neuronal “bad actors” (such as overly connected hub cells), but rather by network changes which enable the onset of pathology among a large populations of neurons.

In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways

Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.

TYPE I INTERFERON-MEDIATED NEUROINFLAMMATORY PROGRAM AND SYNAPSE LOSS IN ALZHEIMER’S DISEASE

The cytokine family type I interferon (IFN) is a major innate immune mediator extensively studied in the peripheral immune responses but largely under-investigated in AD. Previously, we established that innate immune cells readily produce IFN in response to amyloid fibrils containing nucleic acids as cofactor. Here, we investigated whether IFN pathway is associated with amyloidosis in AD brain and contributes to neuroinflammation. By systemically characterizing neuroinflammation in multiple murine AD models, we established a comprehensive core AD neuroinflammation profile that includes several key proinflammatory cytokine families, among which IFN pathway is consistently activated. When hippocampal slice culture was stimulated with different forms of amyloid fibrils, nucleic acid-containing amyloid fibrils, but not heparin-containing fibrils, potently activated IFN pathway and triggered comprehensive neuroinflammation. In addition, stereotaxic administration of IFNβ induced an immune response in the brain of wild type mice analogous to the core neuroinflammatory profile associated with Aβ pathology; whereas selective IFN receptor blockade significantly blunted the ongoing microgliosis in AD models in vivo. Furthermore, IFN promoted microglia-mediated synapse uptake from neurons, which depended on the induction of complement C3, and blockade of IFN signaling significantly abolished the pathogenic synapse loss in AD brain. Consistent with the findings in mice, we found that genes stimulated by IFN were grossly upregulated in human AD brains. Therefore, type I interferon constitutes a major pathway within the neuroinflammatory network of AD and may represent a molecular target to restrain the pathogenic inflammatory responses.

Microglia-dependent presynaptic disruption in an organotypic hippocampal slice culture model of neuroinflammation

Background: Systemic inflammation, such as occurs during sepsis, bone fracture, infections or post-operative trauma, has been linked to synapse loss and cognitive decline in human patients and animal models. Organotypic hippocampal slice cultures (OHSCs) represent an underused tool in neuroinflammation; retaining much of the neuronal architecture, synaptic connections and diversity of cell types present in the hippocampus in vivo whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions as in other in vitro methods. Here we report the development of an OHSC model of synaptic disruption after aseptic inflammation and investigate the underlying mechanism. Methods: OHSCs were generated from P6-P9 C57BL/6, the APP transgenic TgCRND8 model, or wild-type littermate mice according to the interface method. Aseptic inflammation was induced via addition of lipopolysaccharide (LPS) and cultures were analysed for changes in synaptic proteins via western blot. qPCR and ELISA analysis of the slice tissue and culture medium respectively determined changes in gene expression and protein secretion. Microglia were selectively depleted using the toxin clodronate and the effect of IL1β was assessed using a specific neutralising monoclonal antibody. Results: Addition of LPS caused a loss of the presynaptic protein synaptophysin via a mechanism dependent on microglia and involving IL1β. Washout of LPS via medium exchange allows for partial recovery of synaptic protein levels after 2 weeks. TgCRND8 OHSCs do not show alterations in IL1β expression at a timepoint where they exhibit spontaneous synaptophysin loss, and LPS does not alter levels of APP or Aβ in wild-type OHSCs. This indicates that although synaptophysin loss is seen in both systems, there is likely to be distinct underlying pathogenic mechanisms between the neuroinflammatory and amyloid models. Conclusions: We report the development of an OHSC model of LPS-induced synaptophysin loss and demonstrate a key role for microglia and involvement of IL1β. We propose that distinct molecular mechanisms lead to synaptophysin protein loss in LPS- exposed versus TgCRND8 OHSCs and provide a new experimental paradigm for assessing chronic changes in synaptic proteins, and synaptic plasticity, following acute inflammatory insults.

Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high-frequency bursts, faster indicators are required. Here, we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared with iGluSnFR, iGluu has a sixfold faster dissociation rate in vitro and fivefold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100-Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high-frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.