Distinct effects of volatile and intravenous anaesthetics on presynaptic calcium dynamics in mouse hippocampal GABAergic neurones.

BACKGROUND: General anaesthetics have marked effects on synaptic transmission, but their neuronal and circuit-level effects remain unclear. The volatile anaesthetic isoflurane differentially inhibits synaptic vesicle exocytosis in specific neuronal subtypes, but whether other common anaesthetics also have neurone-subtype-specific actions is unknown. METHODS: We used the genetically encoded fluorescent Ca2+ sensor GCaMP6f to compare the pharmacological effects of isoflurane, sevoflurane, propofol, and ketamine on presynaptic excitability in hippocampal glutamatergic neurones and in hippocampal parvalbumin-, somatostatin-, and vasoactive intestinal peptide-expressing (PV+, SST+, and VIP+, respectively) GABAergic interneurones. RESULTS: Isoflurane and sevoflurane depressed activity-driven presynaptic Ca2+ transients in a neurone-type-specific manner, with greater potency for inhibition of glutamate and SST+ compared with PV+ and VIP+ neurone presynaptic activation. In contrast, clinical concentrations of propofol (1 µM) or ketamine (15 µM) had no significant effects on presynaptic activation. Propofol potentiated evoked Ca2+ entry in PV+ interneurones but only at a supraclinical concentration (3 µM). CONCLUSIONS: Anaesthetic-agent-selective effects on presynaptic Ca2+ entry have functional implications for hippocampal circuit function during i.v. or volatile anaesthetic-mediated anaesthesia. Hippocampal interneurones have distinct subtype-specific sensitivities to volatile anaesthetic actions on presynaptic Ca2+, which are similar between isoflurane and sevoflurane.

Selective inhibition of gamma aminobutyric acid release from mouse hippocampal interneurone subtypes by the volatile anaesthetic isoflurane.

BACKGROUND: The cellular and molecular mechanisms by which general anaesthesia occurs is poorly understood. Hippocampal interneurone subpopulations, which are critical regulators of cognitive function, have diverse neurophysiological and synaptic properties, but their responses to anaesthetics are unclear. METHODS: We used live-cell imaging of fluorescent biosensors expressed in mouse hippocampal neurones to delineate interneurone subtype-specific effects of isoflurane on synaptic vesicle exocytosis. The role of voltage-gated sodium channel (Nav) subtype expression in determining isoflurane sensitivity was probed by overexpression or knockdown of specific Nav subtypes in identified interneurones. RESULTS: Clinically relevant concentrations of isoflurane differentially inhibited synaptic vesicle exocytosis: to 83.1% (11.7%) of control in parvalbumin-expressing interneurones, and to 58.6% (13.3%) and 64.5% (8.5%) of control in somatostatin-expressing interneurones and glutamatergic neurones, respectively. The relative expression of Nav1.1 (associated with lower sensitivity) and Nav1.6 (associated with higher sensitivity) determined the sensitivity of exocytosis to isoflurane. CONCLUSIONS: Isoflurane inhibits synaptic vesicle exocytosis from hippocampal glutamatergic neurones and GABAergic interneurones in a cell-type-specific manner depending on their expression of voltage-gated sodium channel subtypes.

Chronic Calcineurin Inhibition via Cyclosporine A Impairs Visuospatial Learning After Isoflurane Anesthesia.

BACKGROUND: Clinical studies implicate the perioperative period in cognitive complications, and increasing experimental evidence shows that the anesthetic agents can affect neuronal processes that underpin learning and memory. Calcineurin, a Ca-dependent phosphatase critically involved in synaptic plasticity, is activated after isoflurane exposure, but its role in the neurological response to anesthesia is unclear. METHODS: We investigated the effect of chronic calcineurin inhibition on postanesthetic cognitive function. Mice were treated with 30 minutes of isoflurane anesthesia during a chronic cyclosporine A regimen. Behavioral end points during the perianesthesia period were quantified. Visuospatial learning was assessed with the water radial arm maze. Total and biotinylated surface protein expression of the α5ß3γ2 γ-aminobutyric acid (GABA) type A receptors was measured. Expression of the GABA synthesis enzyme glutamate decarboxylase (GAD)-67 was also measured. RESULTS: Mice treated with cyclosporine A before anesthesia showed significant deficits in visuospatial learning compared to sham and cyclosporine A-treated mice (n = 10 per group, P = .0152, Tukey post hoc test). Induction and emergence were unaltered by cyclosporine A. Analysis of hippocampal protein expression revealed an increased surface expression of the α5 GABA type A receptor subunit after isoflurane treatment (P = .019, Dunnett post hoc testing), as well as a decrease in GAD-67 expression. Cyclosporine A did not rescue either effect. CONCLUSIONS: Our results confirm the work of others that isoflurane induces changes to inhibitory network function and exclude calcineurin inhibition via cyclosporine A as an intervention. Further, our studies suggest that calcineurin mediates a protective role in the neurological response to anesthesia, and patients receiving cyclosporine A may be an at-risk group for memory problems related to anesthesia.

In vitro and Ex vivo Neurotoxic Effects of Efavirenz are Greater than Those of Other Common Antiretrovirals.

Although antiretroviral (ARV) therapy has reduced the incidence of severe dementia associated with HIV infection, there has been a rise in milder neurocognitive complaints. Data from HIV patients taking ARVs have shown measurable neurocognitive improvements during drug cessation, suggesting a neurotoxic role of the therapy itself. Mechanisms underlying potential ARV neurotoxicity have not been thoroughly investigated, however pathologic oxidative stress and mitochondrial dysfunction have been suspected. Using DIV 16 primary rat cortical neuron culture, we tested eight ARVs from the three most commonly prescribed ARV classes: nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) for effects on neuron viability and morphology after 24 h of drug exposure. Of the tested NRTIs, only stavudine at nearly 100 times the target plasma concentration affected neuron viability with no appreciable change in morphology. Dideoxyinosine induced dendritic simplification at 100 times target plasma concentrations, but did not adversely affect viability. The sole NtRTI, tenofovir, induced dendritic simplification at approximately 3-4 times target plasma concentration, but did not affect viability. Of the tested PIs, only amprenavir decreased neuron viability at nearly 100 times the target plasma concentration. The non-nucleoside reverse transcriptase inhibitor, efavirenz, consistently reduced viability (at 50 µM) and induced dendritic simplification (at 20 µM) nearest the target plasma concentration. Probing mitochondrial energetics of DIV16 cortical neurons after exposure to 20 µM efavirenz showed rapid diminution of mitochondrial-dependent oxygen consumption. Further, 20 µM efavirenz decreased excitability in ex vivo slice culture whereas 2 µM had no effect.

Effects of γ-Aminobutyric Acid Type A Receptor Modulation by Flumazenil on Emergence from General Anesthesia.

BACKGROUND: Transitions into conscious states are partially mediated by inactivation of sleep networks and activation of arousal networks. Pharmacologic hastening of emergence from general anesthesia has largely focused on activating subcortical monoaminergic networks, with little attention on antagonizing the γ-aminobutyric acid type A receptor (GABAAR). As the GABAAR mediates the clinical effects of many common general anesthetics, the authors hypothesized that negative GABAAR modulators would hasten emergence, possibly via cortical networks involved in sleep. METHODS: The authors investigated the capacity of the benzodiazepine rescue agent, flumazenil, which had been recently shown to promote wakefulness in hypersomnia patients, to alter emergence. Using an in vivo rodent model and an in vitro GABAAR heterologous expression system, they measured flumazenil's effects on behavioral, neurophysiologic, and electrophysiologic correlates of emergence from isoflurane anesthesia. RESULTS: Animals administered intravenous flumazenil (0.4 mg/kg, n = 8) exhibited hastened emergence compared to saline-treated animals (n = 8) at cessation of isoflurane anesthesia. Wake-like electroencephalographic patterns occurred sooner and exhibited more high-frequency electroencephalography power after flumazenil administration (median latency ± median absolute deviation: 290 ± 34 s) compared to saline administration (473 ± 186 s; P = 0.042). Moreover, in flumazenil-treated animals, there was a decreased impact on postanesthesia sleep. In vitro experiments in human embryonic kidney-293T cells demonstrated that flumazenil inhibited isoflurane-mediated GABA current enhancement (n = 34 cells, 88.7 ± 2.42% potentiation at 3 µM). Moreover, flumazenil exhibited weak agonist activity on the GABAAR (n = 10 cells, 10.3 ± 3.96% peak GABA EC20 current at 1 µM). CONCLUSIONS: Flumazenil can modulate emergence from isoflurane anesthesia. The authors highlight the complex role GABAARs play in mediating consciousness and provide mechanistic links between emergence from anesthesia and arousal.

Small molecule antagonist reveals seizure-induced mediation of neuronal injury by prostaglandin E2 receptor subtype EP2.

With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE(2)) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists. The 12 most potent compounds displayed competitive antagonism of the human EP2 receptor with K(B) 2-20 nM in Schild regression analysis and 268- to 4,730-fold selectivity over the prostaglandin EP4 receptor. A brain-permeant compound completely suppressed the up-regulation of COX-2 mRNA in rat cultured microglia by EP2 activation and significantly reduced neuronal injury in hippocampus when administered in mice beginning 1 h after termination of pilocarpine-induced status epilepticus. The salutary actions of this novel group of antagonists raise the possibility that selective block of EP2 signaling via small molecules can be an innovative therapeutic strategy for inflammation-related brain injury.

Isoflurane Alters Presynaptic Endoplasmic Reticulum Calcium Dynamics in Wild-Type and Malignant Hyperthermia-Susceptible Rodent Hippocampal Neurons.

Volatile anesthetics reduce excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms which include inhibition of depolarization-evoked increases in presynaptic Ca2+ concentration and blockade of postsynaptic excitatory glutamate receptors. The presynaptic sites of action leading to reduced electrically evoked increases in presynaptic Ca2+ concentration and Ca2+-dependent exocytosis are unknown. Endoplasmic reticulum (ER) of Ca2+ release via ryanodine receptor 1 (RyR1) and uptake by SERCA are essential for regulation intracellular Ca2+ and are potential targets for anesthetic action. Mutations in sarcoplasmic reticulum (SR) release channels mediate volatile anesthetic-induced malignant hyperthermia (MH), a potentially fatal pharmacogenetic condition characterized by unregulated Ca2+ release and muscle hypermetabolism. However, the impact of MH mutations on neuronal function are unknown. We used primary cultures of postnatal hippocampal neurons to analyze volatile anesthetic-induced changes in ER Ca2+ dynamics using a genetically encoded ER-targeted fluorescent Ca2+ sensor in both rat and mouse wild-type (WT) neurons and in mouse mutant neurons harboring the RYR1 T4826I MH-susceptibility mutation. The volatile anesthetic isoflurane reduced both baseline and electrical stimulation-evoked increases in ER Ca2+ concentration in neurons independent of its depression of presynaptic cytoplasmic Ca2+ concentrations. Isoflurane and sevoflurane, but not propofol, depressed depolarization-evoked increases in ER Ca2+ concentration significantly more in mouse RYR1 T4826I mutant neurons than in wild-type neurons. The RYR1 T4826I mutant neurons also showed markedly greater isoflurane-induced reductions in presynaptic cytosolic Ca2+ concentration and synaptic vesicle (SV) exocytosis. These findings implicate RyR1 as a molecular target for the effects of isoflurane on presynaptic Ca2+ handling.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons.

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro-l-arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)-l-ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons.

Relevance of Cortical and Hippocampal Interneuron Functional Diversity to General Anesthetic Mechanisms: A Narrative Review.

General anesthetics disrupt brain processes involved in consciousness by altering synaptic patterns of excitation and inhibition. In the cerebral cortex and hippocampus, GABAergic inhibition is largely mediated by inhibitory interneurons, a heterogeneous group of specialized neuronal subtypes that form characteristic microcircuits with excitatory neurons. Distinct interneuron subtypes regulate specific excitatory neuron networks during normal behavior, but how these interneuron subtypes are affected by general anesthetics is unclear. This narrative review summarizes current principles of the synaptic architecture of cortical and interneuron subtypes, their contributions to different forms of inhibition, and their roles in distinct neuronal microcircuits. The molecular and cellular targets in these circuits that are sensitive to anesthetics are reviewed in the context of how anesthetics impact interneuron function in a subtype-specific manner. The implications of this functional interneuron diversity for mechanisms of anesthesia are discussed, as are their implications for anesthetic-induced changes in neural plasticity and overall brain function.

The Influence of Regional Distribution and Pharmacologic Specificity of GABAAR Subtype Expression on Anesthesia and Emergence.

Anesthetics produce unconsciousness by modulating ion channels that control neuronal excitability. Research has shown that specific GABAA receptor (GABAAR) subtypes in particular regions of the central nervous system contribute to different hyperpolarizing conductances, and behaviorally to distinct components of the anesthetized state. The expression of these receptors on the neuron cell surface, and thus the strength of inhibitory neurotransmission, is dynamically regulated by intracellular trafficking mechanisms. Pharmacologic or activity-based perturbations to these regulatory systems have been implicated in pathology of several neurological conditions, and can alter the individual response to anesthesia. Furthermore, studies are beginning to uncover how anesthetic exposure itself elicits enduring changes in subcellular physiology, including the processes that regulate ion channel trafficking. Here, we review the mechanisms that determine GABAAR surface expression, and elaborate on influences germane to anesthesia and emergence. We address known trafficking differences between the intrasynaptic receptors that mediate phasic current and the extra-synaptic receptors mediating tonic current. We also describe neurophysiologic consequences and network-level abnormalities in brain function that result from receptor trafficking aberrations. We hypothesize that the relationship between commonly used anesthetic agents and GABAAR surface expression has direct consequences on mature functioning neural networks and by extension ultimately influence the outcome of patients that undergo general anesthesia. Rational design of new anesthetics, anesthetic techniques, EEG-based monitoring strategies, or emergence treatments will need to take these effects into consideration.

Force-sensitive afferents recruited during stance encode sensory depression in the contralateral swinging limb during locomotion.

Afferent feedback alters muscle activity during locomotion and must be tightly controlled. As primary afferent depolarization-induced presynaptic inhibition (PAD-PSI) regulates afferent signaling, we investigated hindlimb PAD-PSI during locomotion in an in vitro rat spinal cord-hindlimb preparation. We compared the relation of PAD-PSI, measured as dorsal root potentials (DRPs), to observed ipsilateral and contralateral limb endpoint forces. Afferents activated during stance-phase force strongly and proportionately influenced DRP magnitude in the swinging limb. Responses increased with locomotor frequency. Electrical stimulation of contralateral afferents also preferentially evoked DRPs in the opposite limb during swing (flexion). Nerve lesioning, in conjunction with kinematic results, support a prominent contribution from toe Golgi tendon organ afferents. Thus, force-dependent afferent feedback during stance binds interlimb sensorimotor state to a proportional PAD-PSI in the swinging limb, presumably to optimize interlimb coordination. These results complement known actions of ipsilateral afferents on PAD-PSI during locomotion.