CB1 Cannabinoid Receptor Expression in the Barrel Field Region Is Associated with Mouse Learning

We found previously that fear conditioning by combined stimulation of a row B facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) leads to expansion of the cortical representation of the “trained” row, labeled with 2-deoxyglucose (2DG), in the layer IIIb/IV of the adult mouse the primary somatosensory cortex (S1) 24 h later. We have observed that these learning-dependent plastic changes are manifested by increased expression of somatostatin, cholecystokinin (SST+, CCK+) but not parvalbumin (PV+) immunopositive interneurons We have expanded this research and quantified a numerical value of CB1-expressing and PV-expressing GABAergic axon terminals (CB1+ and PV+ immunopositive puncta) that innervate different segments of postsynaptic cells in the barrel hollows of S1 cortex. We used 3D microscopy to identify the CB+ and PV+ puncta in the barrel cortex “trained” and the control hemispheres CS+UCS group and in controls: Pseudoconditioned, CS-only, UCS-only, and naive animals. We have identified that (i) the association between whisker-shock “trained” barrel B hollows and CB1+, but not PV+ puncta expression remained significant after Bonferroni correction, (ii) CS+UCS has had a significant increasing effect on expression of CB1+ but not PV+ puncta in barrel cortex “trained” hemisphere, and (iii) the pseudoconditioning had a significant decreasing effect on expression of CB1+, but not on PV+ puncta in barrel cortex, both trained and untrained hemispheres. It is correlated to disturbing behaviors. The results suggest that CB1+ puncta regulation is specifically linked with mechanisms leading to learning-dependent plasticity in S1 cortex

Plasma membrane potential of AOB (row A), NOB (row B), visualized by JC-1 (8 µM) in the presence of cyanide (100

<p> <b>µM), azide (75 mM), dicumarol (25 µM), quinacrine (1 mM) and control without xenobiotics.</b> Observation started after 30 min. preincubation in the eppendorf with coverslip. Ammonium concentration 4 mM, nitrite concentration 1.5 mM, temp 20±1°C, pH 7.5±0.1.</p

Relative decrease of NAD(P)H (with respect to the control) in AOB and NOB cells in the presence of cyanide (2.0 µM), azide (1.5 mM), dicumarol (80 µM) and quinacrine (0.5 mM), relative to the control without inhibitors.

<p>Statistically significant differences are indicated with asterisk. Ammonium concentration (AOB) 4 mM, nitrite concentration (NOB) 1.5 mM, temp 20±1°C, pH 7.5±0.1.</p

Hypothetical schematic of NOB ETCh with marked places of electron flow inhibition where a - quinacrine; b - dicumarol; c - azide and cyanide.

<p>Red arrow shown electron flow from NXR (nitrite oxidase) to oxygen; yellow arrow - reverse electron flow from NXR to NAD(P)⁺; blue arrow- reverse proton flow. The model was based on the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053484#pone.0053484-Ferguson1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053484#pone.0053484-Starkenburg1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053484#pone.0053484-Starkenburg2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053484#pone.0053484-Spieck1" target="_blank">[47]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053484#pone.0053484-Lcker1" target="_blank">[51]</a>.</p

Generic composition, spatial architecture and metabolic activity of studied microorganisms consortia.

<p>A – Population composition of bacterial consortia (measured with relative abundancy of specific 16S rDNA genes). B <b>–</b> Microscope image of consortia of nitrifying bacteria: AOB (red) and NOB (green), labeled using FISH. C – Metabolic activity of the consortia measured in the presence of ammonium, nitrite, glucose and acetate. Biomass 0.675 mg mL⁻¹, concentration of ammonium 4 mM, nitrite 1.5 mM, glucose 2.8 mM, acetate 8.3 mM, temp 20±1°C, pH 7.5±0.1.</p

Change of oxygen consumption rate in the presence of cyanide (A), azide (B), dicumarol (C) and quinacrine (D).

<p>Ammonium concentration 4 mM (AOB), nitrite concentration 1.5 mM (NOB), temp 20±1°C, pH 7.5±0.1.</p

Single-cell imaging of the heat-shock response in colon cancer cells suggests that magnitude and length rather than time of onset determines resistance to apoptosis.

Targeting the proteasome is a valuable approach for cancer therapy, potentially limited by pro-survival pathways that are induced in parallel to cell death. Whether these pro-survival pathways are activated in all cells, show different activation kinetics in sensitive versus resistant cells or interact functionally with cell death pathways is unknown. We monitored activation of the heat-shock response (HSR), a key survival pathway induced by proteasome inhibition, relative to apoptosis activation in HCT116 colon cancer cells expressing enhanced green fluorescent protein (EGFP) under the control of the HSP70 promoter. Single-cell and high-content time-lapse imaging of epoxomicin treatment revealed that neither basal activity nor the time of onset of the HSR differed between resistant and sensitive populations. However, resistant cells had significantly higher and prolonged reporter activity than those that succumbed to cell death. p53 deficiency protected against cell death but failed to modulate the HSR. By contrast, inhibition of the HSR significantly increased the cytotoxicity of epoxomicin. Our data provide novel insights into the kinetics and heterogeneity of the HSR during proteasome inhibition, suggesting that the HSR modulates cell death signalling unidirectionally.</p

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

<div><p>Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca²⁺ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.</p></div

Pharmacological inhibition of cell death pathways protects neurons against glutamate-induced cell death.

<p>Neurons were seeded at 50,000 cells/well and cultured <i>in vitro</i> for 7/8 days. Neurons were pre-treated for 1h prior to glutamate excitation with calpeptin (20 nM), rapamycin (250 nM), DPQ (100 μM), Compound C (10 μM) or SP600125 (5 μM). Following glutamate exposure (Glut.; 100 μM for 10 or 30 min,), media was replaced with pre-conditioned media alone (in the case of Compound C and SP600125) or pre-conditioned media containing the drugs, and images were acquired for 24 h. A) Populations (Popul.) of neurons classified as viable (blue traces), apoptotic (green traces) and necrotic (red traces). Traces show the median ± inter-quartile regions (n = 4 wells from 2 independent experiments). Traces show that blocking key cell death signalling pathways protected neurons against excitotoxicity. B) A heat map of the median population of viable cells 24 h following glutamate excitation (100 μM) illustrates the protective effect of these drugs against glutamate-induced cell death. C) Boxplots showing % of viable, apoptotic and necrotic neurons 24 h following 100 μM glutamate excitation for 10 min. Boxplots show the median ± inter-quartile regions. Pre-treatment with Calpeptin, DPQ and SP 600125 significantly increased viability compared to no pre-treatment and this increased viability was primarily due to a decrease in apoptosis (*p = 0.0475 for all marked treatments compared to no pre-treatment).</p

CGNs seeded either at low or high seeding densities undergo increased necrosis in response to glutamate excitation.

<p>Cerebellar granule neurons (CGNs) seeded at 25,000, 50,000 or 100,000 cells/well were cultured for 7/8 DIV and treated with 10, 30 or 100 μM glutamate (Glut; with 10 μM glycine) for 10 min before media was replaced with high Mg²⁺ (1.2 mM) containing buffer to block glutamate receptors. For 24 h following glutamate exposure, 9 fields of view per well (550–650 cells/field of view) were imaged at 1 hr intervals, and neurons were classified as viable, apoptotic or necrotic based on their nuclear morphology (Hoechst) and plasma membrane integrity (PI). A) Representative transmitted light images of CGNs seeded at 25,000, 50,000 or 100,000 cells/well in 96-well plates and cultured <i>in vitro</i> for 7 days. Scale bar 40 μm. B) The population of cells (%), for each density and glutamate treatment, classified as viable (blue traces), apoptotic (green traces) or necrotic (red traces). Traces are median ± inter-quartile regions of all wells exposed to the same treatment. C) Quantification of viable, apoptotic and necrotic populations 24 h following glutamate exposure for each of the seeding densities and glutamate concentrations. Boxplots show the median ± inter-quartile regions (n = 8 wells for each treatment, from 4 independent experiments). Neurons seeded at 50,000 cells/well had lower well-to-well variability than neurons seeded at 25,000 or 100,000 cells/well, and underwent less necrotic cell death in response to glutamate excitotoxicity. Neurons seeded at 50,000 cells/well and treated with 100 μM glutamate underwent significantly increased apoptosis compared to neurons treated with 10 μM glutamate (*p = 0.014).</p