Anesthetic Sevoflurane Causes Rho-Dependent Filopodial Shortening in Mouse Neurons

Abstract

Early postnatal anesthesia causes long-lasting learning and memory impairment in rodents, however, evidence for a specific neurotoxic effect on early synaptogenesis has not been demonstrated. Drebrin A is an actin binding protein whose localization in dendritic protrusions serves an important role in dendritic spine morphogenesis, and is a marker for early synaptogenesis. We therefore set out to investigate whether clinically-relevant concentrations of anesthetic sevoflurane, widely- used in infants and children, alters dendritic morphology in cultured fetal day 16 mouse hippocampal neurons. After 7 days in vitro, mouse hippocampal neurons were exposed to four hours of 3% sevoflurane in 95% air/5% CO2 or control condition (95% air/5% CO2). Neurons were fixed in 4% paraformaldehyde and stained with Alexa Fluor555-Phalloidin, and/or rabbit anti-mouse drebrin A/E antibodies which permitted subcellular localization of filamentous (F)-actin and/or drebrin immunoreactivity, respectively. Sevoflurane caused acute significant length-shortening in filopodia and thin dendritic spines in days-in-vitro 7 neurons, an effect which was completely rescued by co-incubating neurons with ten micromolar concentrations of the selective Rho kinase inhibitor Y27632. Filopodia and thin spine recovered in length two days after sevoflurane exposure. Yet cluster-type filopodia (a precursor to synaptic filopodia) were persistently significantly decreased in number on day-in-vitro 9, in part owing to preferential localization of drebrin immunoreactivity to dendritic shafts versus filopodial stalks. These data suggest that sevoflurane induces F-actin depolymerization leading to acute, reversible length-shortening in dendritic protrusions through a mechanism involving (in part) activation of RhoA/Rho kinase signaling and impairs localization of drebrin A to filopodia required for early excitatory synapse formation.

Resveratrol Mitigates Sevoflurane-Induced Neurotoxicity by the SIRT1-Dependent Regulation of BDNF Expression in Developing Mice

“…In particular, synapsin1, vesicular glutamate transporters (VGluT1), PSD95, and Homer1a are classical markers of presynaptic and postsynaptic proteins, which play an important role in synaptic activity [54][55][56][57][58][59]. Previous research has reported that the repeated sevoflurane exposure downregulates synaptic proteins such as postsynaptic protein PSD95 in the hippocampus of developing mice [60,61]. In the present study, we detect the effect of sevoflurane and resveratrol on the expression of these proteins.…”

Ketamine destabilizes growth of dendritic spines in developing hippocampal neurons in�vitro via a Rho‑dependent mechanism

“…A previous study demonstrated that the anesthetic agent isoflurane destabilizes spine actin, which results in alterations to dendritic spine morphology and number ( 31 ). Zimering et al ( 17 ) additionally observed that sevoflurane causes actin depolymerization, leading to reversible length-shortening of dendritic protrusions through a mechanism involving activation of RhoA/ROCK signaling. Ketamine is commonly used as an intravenous general anesthetic for pediatric patients.…”

“…The present study has a number of limitations. Exposure to anesthetics transiently affects the dynamics of dendritic spines, and dendritic protrusion loss and shrinkage are reversible ( 17 , 31 , 38 ). The present study was based on single time-point observations; it is not known whether ketamine-induced effects on the morphology and number of dendritic spines may be attenuated upon ketamine elimination.…”

“…Fluorescence images were captured using confocal microscopy (magnification, ×400 and ×1,000; Nikon Corporation). Analysis of spine plasticity was performed using ImageJ software, as previously described ( 17 ). Dendrite segments located distal to the first branch point from the cell soma and including the most distally-located dendritic segments (2–5 per neuron) were randomly selected for analysis.…”

“…Transforming protein RhoA (RhoA) guanosine triphosphate hydrolases (GTPases) and their downstream signals are critical in the association of neuronal activity with various forms of neuronal plasticity ( 14 , 15 ). Volatile anesthetics lead to neuronal apoptosis and length-shortening of dendritic protrusions by activating RhoA in developing neurons ( 16 , 17 ). In the present study, it was hypothesized that ketamine may alter the number and length of dendritic spines in early developing hippocampal neurons via activation of the RhoA/Rho-associated kinase (ROCK) signaling pathway.…”