Ketamine Reduces the Surface Density of the Astroglial Kir4.1 Channel and Inhibits Voltage-Activated Currents in a Manner Similar to the Action of Ba2+ on K+ Currents.

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression. We examined how ketamine affects inwardly rectifying K+ channel Kir4.1, the principal regulator of K+ buffering and neuronal excitability in the brain. Cultured rat cortical astrocytes were transfected with plasmid-encoding fluorescently tagged Kir4.1 (Kir4.1-EGFP) to monitor the mobility of Kir4.1-EGFP vesicles at rest and after ketamine treatment (2.5 or 25 µM). Short-term (30 min) ketamine treatment reduced the mobility of Kir4.1-EGFP vesicles compared with the vehicle-treated controls (p < 0.05). Astrocyte treatment (24 h) with dbcAMP (dibutyryl cyclic adenosine 5'-monophosphate, 1 mM) or [K+]o (15 mM), which increases intracellular cAMP, mimicked the ketamine-evoked reduction of mobility. Live cell immunolabelling and patch-clamp measurements in cultured mouse astrocytes revealed that short-term ketamine treatment reduced the surface density of Kir4.1 and inhibited voltage-activated currents similar to Ba2+ (300 µM), a Kir4.1 blocker. Thus, ketamine attenuates Kir4.1 vesicle mobility, likely via a cAMP-dependent mechanism, reduces Kir4.1 surface density, and inhibits voltage-activated currents similar to Ba2+, known to block Kir4.1 channels.

Astrocyte arborization enhances Ca2+ but not cAMP signaling plasticity.

The plasticity of astrocytes is fundamental for their principal function, maintaining homeostasis of the central nervous system throughout life, and is associated with diverse exposomal challenges. Here, we used cultured astrocytes to investigate at subcellular level basic cell processes under controlled environmental conditions. We compared astroglial functional and signaling plasticity in standard serum-containing growth medium, a condition mimicking pathologic conditions, and in medium without serum, favoring the acquisition of arborized morphology. Using opto-/electrophysiologic techniques, we examined cell viability, expression of astroglial markers, vesicle dynamics, and cytosolic Ca2+ and cAMP signaling. The results revealed altered vesicle dynamics in arborized astrocytes that was associated with increased resting [Ca2+ ]i and increased subcellular heterogeneity in [Ca2+ ]i , whereas [cAMP]i subcellular dynamics remained stable in both cultures, indicating that cAMP signaling is less prone to plastic remodeling than Ca2+ signaling, possibly also in in vivo contexts.

Astroglial Mechanisms of Ketamine Action Include Reduced Mobility of Kir4.1-Carrying Vesicles.

The finding that ketamine, an anaesthetic, can elicit a rapid antidepressant effect at low doses that lasts for weeks in patients with depression is arguably a major achievement in psychiatry in the last decades. However, the mechanisms of action are unclear. The glutamatergic hypothesis of ketamine action posits that ketamine is a N-methyl-D-aspartate receptor (NMDAR) antagonist modulating downstream cytoplasmic events in neurons. In addition to targeting NMDARs in synaptic transmission, ketamine may modulate the function of astroglia, key homeostasis-providing cells in the central nervous system, also playing a role in many neurologic diseases including depression, which affects to 20% of the population globally. We first review studies on astroglia revealing that (sub)anaesthetic doses of ketamine attenuate stimulus-evoked calcium signalling, a process of astroglial cytoplasmic excitability, regulating the exocytotic release of gliosignalling molecules. Then we address how ketamine alters the fusion pore activity of secretory vesicles, and how ketamine affects extracellular glutamate and K+ homeostasis, both considered pivotal in depression. Finally, we also provide evidence indicating reduced cytoplasmic mobility of astroglial vesicles carrying the inward rectifying potassium channel (Kir4.1), which may regulate the density of Kir4.1 at the plasma membrane. These results indicate that the astroglial capacity to control extracellular K+ concentration may be altered by ketamine and thus indirectly affect the action potential firing of neurons, as is the case in lateral habenula in a rat disease model of depression. Hence, ketamine-altered functions of astroglia extend beyond neuronal NMDAR antagonism and provide a basis for its antidepressant action through glia.

Thermal tolerance of E. faecalis to pulsed heating in the millisecond range.

Our aim was to evaluate thermal damage to endodontic pathogen Enterococcus faecalis (E. faecalis) caused by sub-second laser-generated heat pulses by determining the parameters for the thermal damage survival curve (TDSC). A novel experimental method for thermal pulsing of bacteria in the millisecond range was developed. After cultivation, E. faecalis was inoculated on anodized aluminum substrate and heated with a pulsed Nd:YAG laser. Viability was assessed with both plate count and flow cytometry methods. An E. faecalis TDSC for single-pulse millisecond range heating times was derived from the Arrhenius equation. Results gained from single-pulse heating viability measurements were used to predict the bactericidal effect of multiple sequential pulses (pulse train), and compared to experimental measurements. The thermal damage model was then applied to determine the relationship between laser fluence, pulse width, and the viability decrease of E. faecalis in a simulated root canal disinfection procedure. The application of the model to calculate the required lethal laser fluence levels on dentin during endodontic laser procedures seems to indicate that for endodontic procedures, the sub-millisecond pulsed Nd:YAG lasers are more effective in comparison with continuous-mode diode lasers and will cause less undesirable bulk heating of the tooth and surrounding tissues. The results of the study can be applied to create a model for predicting the impact of sub-second temperature increase on viability of bacteria on various surfaces and calculate required fluences and pulse widths to achieve the aforementioned effects with laser pulses.

Study of the direct bactericidal effect of Nd:YAG and diode laser parameters used in endodontics on pigmented and nonpigmented bacteria.

Laser light can be used during endodontic procedures to sterilize the root canal by destroying bacteria. Previous in-vitro studies that investigated the mechanism of the destruction of bacteria inhabiting the root canal by 1,064-nm Nd:YAG and 808-nm diode laser light used substrates that absorb light in the near-infrared (NIR) spectrum. These substrates heat the bacterial microenvironment, which possibly contributes to cell death. To determine the direct effect of laser light on the bacterial sample in the absence of detrimental heating, a sapphire substrate, which is virtually transparent in NIR spectrum, was inoculated with bacterial samples and subjected to laser irradiation at 1,064 nm (1.5 W, 15 Hz) and at 808 nm (1.5 W, 20 Hz). Enterococcus faecalis, Escherichia coli, and Porphyromonas gingivalis bacteria were used. E. faecalis and E. coli were largely unaffected by laser light. The viability of P. gingivalis, a pigmented bacterium, was directly affected by both NIR wavelengths (a 57% decrease of viability at 1,064 nm and a 31% decrease at 808 nm). Our results indicate that the primary mediator of cell death appears to be the interaction between NIR laser light and the bacterial microenvironment, most likely in the form of heating. Our research suggests that when optimizing the efficacy of laser-assisted endodontic sterilization of the root canal, the optical characteristics of the bacterial microenvironment play a key role, as nonpigmented bacteria appear to be virtually transparent at 808 nm and 1,064 nm.