Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions

Hupp, Sabrina; Tomov, Nikola; Bischoff, C. A.; Baronti, Dario; Iliev, Alexandar

doi:10.1016/j.jneumeth.2021.109405

Cited by 5 publications

(6 citation statements)

References 22 publications

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“…Prior to recording slices were incubated for 60 min at room temperature in either a control-protein or active-protein solutions. The incubation system we used is like the one described by Hupp et al [ 45 ]. We used 12 well-standard culture plates with a strainer basket inside.…”

Section: Methodsmentioning

confidence: 99%

TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

et al. 2023

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Background TREM2 is a transmembrane receptor expressed by myeloid cells and acts to regulate their immune response. TREM2 governs the response of microglia to amyloid and tau pathologies in the Alzheimer’s disease (AD) brain. TREM2 is also present in a soluble form (sTREM2), and its CSF levels fluctuate as a function of AD progression. Analysis of stroke and AD mouse models revealed that sTREM2 proteins bind to neurons, which suggests sTREM2 may act in a non-cell autonomous manner to influence neuronal function. sTREM2 arises from the proteolytic cleavage of the membrane-associated receptor. However, alternatively spliced TREM2 species lacking a transmembrane domain have been postulated to contribute to the pool of sTREM2. Thus, both the source of sTREM2 species and its actions in the brain remain unclear. Methods The expression of TREM2 isoforms in the AD brain was assessed through the analysis of the Accelerating Medicines Partnership for Alzheimer’s Disease Consortium transcriptomics data, as well as qPCR analysis using post-mortem samples of AD patients and of the AD mouse model 5xFAD. TREM2 cleavage and secretion were studied in vitro using HEK-293T and HMC3 cell lines. Synaptic plasticity, as evaluated by induction of LTP in hippocampal brain slices, was employed as a measure of sTREM2 actions. Results Three distinct TREM2 transcripts, namely ENST00000373113 (TREM2230), which encodes the full-length transmembrane receptor, and the alternatively spliced isoforms ENST00000373122 (TREM2222) and ENST00000338469 (TREM2219), are moderately increased in specific brain regions of patients with AD. We provide experimental evidence that TREM2 alternatively spliced isoforms are translated and secreted as sTREM2. Furthermore, our functional analysis reveals that all sTREM2 species inhibit LTP induction, and this effect is abolished by the GABAA receptor antagonist picrotoxin. Conclusions TREM2 transcripts can give rise to a heterogeneous pool of sTREM2 which acts to inhibit LTP. These results provide novel insight into the generation, regulation, and function of sTREM2 which fits into the complex biology of TREM2 and its role in human health and disease. Given that sTREM2 levels are linked to AD pathogenesis and progression, our finding that sTREM2 species interfere with LTP furthers our understanding about the role of TREM2 in AD.

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Section: Methodsmentioning

confidence: 99%

TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

et al. 2023

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“…Hypoxia increased lytic pore formation after 120 min ( Figure 2 A,B). Challenging acute brain slices with 2 HU/mL PLY either in continuous optimal oxygenation (using carbogen (95% O 2 /5% CO 2 ) perfusion) conditions for 8 h [ 22 ] or on a rotary shaker (in a 5% CO 2 environment and normal atmospheric oxygen, leading to hypoxia in the slice cultures) for the same time led to a two-fold increase in the PLY-induced LDH release during hypoxia (another lytic permeabilization loss marker), indicative of the presence of elevated delayed (8 h) hypoxic damage ( Figure 2 C). The challenge with 2 HU/mL PLY, despite the evidence for synaptic loss [ 12 ], did not elevate the LDH release when the slices were adequately oxygenized ( Figure 2 C).…”

Section: Resultsmentioning

confidence: 99%

“…Acute brain slices were prepared from infant (postnatal day 10-14) C57Bl/6 mice or Wistar rats via decapitation and vibratome sectioning (Vibroslice NVSL, World Precision Instruments, Berlin, Germany) in artificial CSF continuously oxygenized with carbogen gas (95% O 2 , 5% CO 2 ) at 4 • C. The slices were then allowed to adapt in carbogenated Basal Medium Eagle (Gibco) with 1% penicillin/streptavidin and 80 mg% glucose at 37 • C for 1 h before being incubated in pure BME supplemented with 80 mg% glucose. In some experiments, the brain slices were oxygenated with carbogen continuously during exposure to the toxin or mock [22]. For experiments in hypoxic conditions, slices were incubated in the same medium on a rotary shaker (60 rpm/min) in a 5% CO 2 incubator, with each slice being individually incubated in a separate well of a 24-well plate (Sarstedt).…”

Section: Cell Culture and Tissue Culturementioning

confidence: 99%

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae

Hoffet,

Tomov,

Hupp

et al. 2024

Toxins

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A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin’s pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

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“…Earlier works that analyzed the effect of pneumococci and their factors on dendritic spines relied on an acute brain slice system using slices dipped in artificial cerebrospinal fluid containing lysates or bacteria ( Hupp et al, 2021 ). While such a system allows multiple treatments in parallel and a reduction in the number of animals used, it presents a major drawback—toxic factors access the tissue from all sides, improving toxic factor penetration.…”

Section: Discussionmentioning

confidence: 99%

“…Acute slices were prepared from the brains of newborn C57Bl/6JRj mice at postnatal day (PD) 10–12, according to a previously published protocol ( Hupp et al, 2021 ). Under isoflurane anesthesia, decapitation, skull opening, and cranial plate removal were performed, and the brain was transferred to PBS.…”

Section: Methodsmentioning

confidence: 99%

Dendritic spine loss deep in the neocortex and dendrite distortion with diffusion disturbances occur early in experimental pneumococcal meningitis

et al. 2023

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IntroductionStreptococcus pneumoniae (pneumococcus) meningitis is a serious disease with substantial lethality and long-term disability in survivors. Loss of synaptic staining in the superficial layers of the neocortex in rodent models and in humans, and pneumolysin (a major pneumococcal toxin)-dependent dendritic spine collapse in brain slices have been described. It remains unclear how deep in the neocortex more discrete changes are present, how soon after disease onset these changes occur, and whether other properties of dendrites are also affected.MethodsUsing a mouse model of pneumococcal meningitis, we studied changes in the neocortex shortly (3–6 h) after the onset of clinical symptoms via modified Golgi-Cox silver staining.ResultsDendritic changes were present in areas with otherwise unchanged cell numbers and no signs of necrosis or other apparent neuronal pathology. Mature dendritic spines were reduced in the pyramidal neurons running through layers 1–5. Additionally, spine morphology changes (swelling, spine neck distortion), were also observed in the deeper layers 4 and 5 of the neocortex. Immature spines (filopodia) remained unchanged between groups, as well as the dendritic arborization of the analyzed neurons. In a third of the animals with meningitis, massive mechanical distortion of the primary dendrites of most of the pyramidal neurons through layers 1–5 was observed. This distortion was reproduced in acute brain slices after exposure to pneumolysin-containing bacterial lysates (S. pneumoniae D39 strain), but not to lysates of pneumolysin-deficient bacteria, which we explain by the tissue remodeling effect of the toxin. Experimental mechanical dendrite distortion in primary neural cultures demonstrated diminished FRAP diffusion of neuronally-expressed enhanced green fluorescent protein (eGFP), indicative of disturbed dendritic diffusion.DiscussionOur work extends earlier knowledge of synaptic loss in the superficial cortical layers during meningitis to deeper layers. These changes occurred surprisingly early in the course of the disease, substantially limiting the effective therapeutic window. Methodologically, we demonstrate that the dendritic spine collapse readout is a highly reliable and early marker of neural damage in pneumococcal meningitis models, allowing for reduction of the total number of animals used per a group due to much lower variation among animals.

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Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions

Cited by 5 publications

References 22 publications

TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

TREM2 splice isoforms generate soluble TREM2 species that disrupt long-term potentiation

Glucose and Oxygen Levels Modulate the Pore-Forming Effects of Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae

Dendritic spine loss deep in the neocortex and dendrite distortion with diffusion disturbances occur early in experimental pneumococcal meningitis

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