Andrey B. Petrenko

Mutation of α1GT-type Calcium Channels in Mice Does Not Change Anesthetic Requirements for Loss of the Righting Reflex and Minimum Alveolar Concentration but Delays the Onset of Anesthetic Induction

Background:T-type calcium channels regulate neuronal membrane excitability and participate in a number of physiologic and pathologic processes in the central nervous system, including sleep and epileptic activity. Volatile anesthetics inhibit native and recombinant T-type calcium channels at concentrations comparable to those required to produce anesthesia. To determine whether T-type calcium channels are involved in the mechanisms of anesthetic action, the authors examined the effects of general anesthetics in mutant mice lacking α1G T-type calcium channels. Methods:The hypnotic effects of volatile and intravenous anesthetics administered to mutant and C57BL/6 control mice were evaluated using the behavioral endpoint of loss of righting reflex. To investigate the immobilizing effects of volatile anesthetics in mice, the minimum alveolar concentration (MAC) values were determined using the tail-clamp method. Results:The 50% effective concentration for loss of righting reflex and MAC values for volatile anesthetics were not altered after α1G channel knockout. However, mutant mice required significantly more time to develop anesthesia/hypnosis after exposure to isoflurane, halothane, and sevoflurane and after intraperitoneal administration of pentobarbital. Conclusions:The 50% effective concentration for loss of righting reflex and MAC values for the volatile anesthetics were not altered after α1G calcium channel knockout, indicating that normal functioning of α1G calcium channels is not required for the maintenance of anesthetic hypnosis and immobility. However, the timely induction of anesthesia/hypnosis by volatile anesthetic agents and some intravenous anesthetic agents may require the normal functioning of these channel subunits.

Reduced Sensitivity to Ketamine and Pentobarbital in Mice Lacking the N-Methyl-d-Aspartate Receptor GluRε1 Subunit

Ketamine is an IV anesthetic with N-methyl-d-aspartate receptor (NMDAR)-blocking properties. However, it is still unclear whether ketamine’s general anesthetic actions are mediated primarily via blockade of NMDAR. Functional NMDARs are composed by the assembly of a GluR&zgr;1 (NR1) subunit with GluR&egr; (GluR&egr;1–4; NR2A–D) subunits, which confer unique properties on native NMDARs. We hypothesized that animals deficient in GluR&egr;1, an abundant and ubiquitously postnatally expressed NMDAR subunit, might be resistant to the effects of ketamine. Here, we evaluated a righting reflex to determine the general anesthetic/hypnotic potency of ketamine administered intraperitoneally to GluR&egr;1 knockout mice and compared these results with those for wild-type mice. Mutant mice were more resistant to ketamine than control mice. Unexpectedly, mutant mice were also more resistant to pentobarbital, which is thought not to interact with NMDAR at clinically relevant concentrations. Although these data in no way eliminate the possibility of the involvement of the NMDAR GluR&egr;1 subunit in mediation of ketamine anesthesia/hypnosis, they suggest the difficulties with interpretation of altered anesthetic sensitivity in knockout animal models.

Taurine activates glycine and γ-aminobutyric acid A receptors in rat substantia gelatinosa neurons

Taurine has been suggested to modulate nociceptive information at the spinal cord level. In this study, the pharmacological properties of taurine were investigated in adult rat substantia gelatinosa (SG) neurons using whole-cell patch-clamp method. We found that taurine seemed to have higher efficacy than glycine on glycine receptors in SG neurons. An increase in chloride conductance was responsible for taurine-induced currents. Taurine at 0.3 mM activated glycine receptors, whereas at 3 mM activated both glycine and &ggr;-aminobutyric acid A receptors. The currents activated by coapplication of taurine and glycine are cross inhibitive. Altogether these results show that taurine might represent another important neurotransmitter or modulator in SG neurons, which may be involved in antinociception.

Defining the role of NMDA receptors in anesthesia: are we there yet?

N-methyl-d-aspartate (NMDA) receptors are important in mediating excitatory neurotransmission in the nervous system. They are preferentially inhibited by some general anesthetics and have, therefore, been implied in the mediation of their effects. This review summarizes the main research findings available related to NMDA receptors and their role in anesthesia. The contribution of NMDA receptors to the anesthetized state is discussed separately for each of its components: amnesia, analgesia, unconsciousness and immobility. Anesthetic-induced unconsciousness and immobility have received the most attention in the research community and are the main focus of this review. In the overall perspective, however, studies using pharmacological or electrophysiological approaches have failed to reach definitive conclusions regarding the contribution of NMDA receptors to these anesthetic endpoints. None of the studies have specifically addressed the role of NMDA receptors in the amnestic effect of general anesthetics, and the few available data are (at best) only indirect. NMDA receptor antagonism by general anesthetics may have a preventive anti-hyperalgesic effect. The only and most extensively used genetic tool to examine the role of NMDA receptors in anesthesia is global knockout of the GluN2A subunit of the NMDA receptor. These animals are resistant to many intravenous and inhalational anesthetics, but the interpretation of their phenotype is hindered by the secondary changes occurring in these animals after GluN2A knockout, which are themselves capable of altering anesthetic sensitivity. Generation of more sophisticated conditional knockout models targeting NMDA receptors is required to finally define their role in the mechanisms of anesthesia.

The NR3B Subunit Does Not Alter the Anesthetic Sensitivities of Recombinant N-Methyl-D-Aspartate Receptors

The N-methyl-d-aspartate (NMDA) receptor NR3B subunit co-assembles with NR1 and NR2 subunits to form a receptor complex with distinct channel properties. In the present study, we investigated the effects of co-expression of the NR3B subunit on the anesthetic sensitivities of NMDA receptors for NR1/NR2 channels expressed in Xenopus oocytes. Although the NR3B subunit prominently reduced the current amplitude of NR1/NR2A-B channels, the sensitivities of NR1/NR2A-B channels to Mg2+, ketamine, isoflurane, nitrous oxide, and ethanol were not altered by co-expression of the NR3B subunit. These results suggest that the anesthetic sensitivities of NMDA receptors do not depend on the presence or absence of the NR3 subunit. Mutations of two amino acid residues in the NR3B subunit at positions homologous to the N and N + 1 sites in the NR1 and NR2 subunits, which constitute the blocking sites for Mg2+ and ketamine, did not affect the sensitivities of NR1/NR2B/NR3B channels to Mg2+, ketamine and isoflurane. Thus, the amino acid residues at the N and N + 1 sites in NR3 subunits are unlikely to be involved in the formation of channel blocking sites in NR1/NR2/NR3 channels.

Effect of agmatine on heteromeric N-methyl-D-aspartate receptor channels

Endogenous polyamines like spermine are known to have four distinct effects on recombinant N-methyl-d-aspartate (NMDA) receptor channels: voltage-dependent inhibition, glycine-dependent stimulation, glycine-independent stimulation and decreased affinity to the agonist (l-glutamate). These effects are highly dependent on the constituting epsilon subunits (epsilon1-epsilon4) of the recombinant NMDA receptor channels. Agmatine reportedly inhibits native NMDA receptor channels in cultured hippocampal neurons. In the present investigation, the effects of agmatine on the epsilon/zeta heteromeric NMDA receptor channels expressed in Xenopus laevis oocytes were examined using the two-electrode voltage clamp method. Agmatine inhibited the four epsilon/zeta (epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1 and epsilon4/zeta1) channels with similar sensitivity (an IC50 value of about 300microM at -70mV). This effect was dependent on the membrane potential and was more pronounced at hyperpolarized membrane potentials (voltage-dependent inhibition). Agmatine did not exhibit other stimulatory (glycine-dependent and -independent effects) or inhibitory (decreased affinity to l-glutamate) effects. These properties are similar to the pharmacological profile of well-characterized NMDA receptor channel blockers like phencyclidine and ketamine. Thus, regarding the effects on the NMDA receptor channels, agmatine is not like other endogenous polyamines rather it acts as a channel blocker.

Effects of ketamine on acute somatic nociception in wild-type and N-methyl-d-aspartate (NMDA) receptor ɛ1 subunit knockout mice

Although the properties of ketamine appear to be well characterized, there is a lot of ambiguity in the literature regarding its analgesic effects. After careful selection of proper experimental conditions and drug doses, we systematically characterized the effects of systemic ketamine on acute somatic nociception in mice and examined the role of the NMDA receptor epsilon1 subunit in mediating its analgesia. Intraperitoneal administration of ketamine was not analgesic in any of the phasic pain assays (thermal, mechanical, electrical) applied to C57BL/6 (wild-type) and NMDA receptor epsilon1 subunit knockout (mutant) mice. Surprisingly, rather than being analgesic for thermal nociception, ketamine showed pronociceptive properties in case of low-intensity heat stimulation in wild-type mice. In the formalin test (tonic pain), ketamine significantly reduced phase 2 nociceptive behavior in both wild-type and mutant mice. These data indicate that in wild-type mice ketamine has no analgesic effect on phasic pain in normal somatic tissues, but alleviates tonic pain after inflammation. Such analgesic spectrum of ketamine can be fully explained by its NMDA receptor antagonist properties. The results for the mutant mice suggest that the epsilon1 subunit of the NMDA receptor does not mediate the analgesic effects of ketamine in tonic pain.

Augmented tonic pain-related behavior in knockout mice lacking monoacylglycerol lipase, a major degrading enzyme for the endocannabinoid 2-arachidonoylglycerol.

Monoacylglycerol lipase (MGL) is the main enzyme responsible for degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective inhibitors of MGL have antinociceptive effects upon acute administration and, therefore, hold promise as analgesics. To gain insight into the possible consequences of their prolonged administration, genetically modified mice with the knocked-out MGL gene were tested in several models of acute (phasic, tonic) and chronic (inflammatory, neuropathic) pain. MGL knockout mice showed normal acute phasic pain perception (pain thresholds) and no alleviation of pain perception in models of inflammatory and neuropathic pain. However, compared with wild-type controls, they showed significantly augmented nociceptive behavior in models of acute somatic and visceral tonic pain (formalin and acetic acid tests). The observed proalgesic changes in perception of tonic pain in MGL knockouts could have resulted from desensitization of cannabinoid receptors (known to occur after genetic inactivation of MGL). Supporting this notion, chronic pretreatment with the selective CB1 receptor antagonist AM 251 (employed to re-sensitize cannabinoid receptors in MGL knockouts) resulted in normalization of their tonic pain-related behaviors. Similar augmentation of tonic pain-related behaviors was replicated in C57BL/6N mice pretreated chronically with the selective MGL inhibitor JZL 184 (employed to pharmacologically desensitize CB1 receptors). These findings imply that prolonged use of MGL inhibitors, at doses causing close to complete inhibition of MGL enzymatic activity, not only have no beneficial analgesic effects, they may lead to exacerbation of some types of pain (particularly those with a tonic component).

No evidence for the development of acute analgesic tolerance during and hyperalgesia after prolonged remifentanil administration in mice

BackgroundAcute opioid tolerance (AOT) and opioid-induced hyperalgesia (OIH) are undesirable effects of opioids that have been reported in both animals and humans. However, the development of AOT and OIH in cases of potent, short-acting μ-opioid receptor agonist remifentanil administration remains controversial. It has been suggested that the emergence of AOT and OIH by remifentanil could be dose and infusion duration dependent, i.e., low dose and short infusions may lead to negative results. In this study, we determined whether AOT and OIH could be elicited by prolonged, continuous administration of remifentanil at maximally tolerable doses in C57BL/6 mice.ResultsThe analgesic effects of continuously administered remifentanil [by short (1 h) and prolonged (4 h) intraperitoneal infusions] were studied. These experiments involved repeated measurements of thermal thresholds during remifentanil administration. Therefore, particular attention was paid to prevent cumulative tissue injury, which could mimic pronociceptive effects of remifentanil. To exclude the possibility of pseudoAOT during infusion, we used brief cooling of all ipsilateral hindpaws that exhibited analgesic response. Thermal thresholds remained steadily elevated over a 1-h period during continuous administration at infusion rates of 120, 180, and 240 mg/kg/h, which indicated no AOT development. To exclude the possibility of pseudoOIH after infusion, intact contralateral hindpaws were used for all postinfusion threshold measurements. Thermal thresholds at each infusion rate returned to the baseline values within 15 min after the termination of the administration. They did not decrease below the baseline values during 1 h following infusion, which indicated no OIH development. Similar threshold dynamics were also observed for thermal and mechanical testing modalities in animals infused at 120 mg/kg/h for 4 h as well as in animals with rapidly attained and maintained maximum analgesia for 3 h.ConclusionsThese results suggest that neither intra-infusion AOT nor postinfusion OIH develops in mice receiving continuous remifentanil when the possibility of cumulative tissue injury mimicking AOT or OIH is carefully avoided.

Reduced Immobilizing Properties of Isoflurane and Nitrous Oxide in Mutant Mice Lacking the N-Methyl-d-Aspartate Receptor GluRε1 Subunit Are Caused by the Secondary Effects of Gene Knockout

BACKGROUND: Until recently, the N-methyl-d-aspartate (NMDA) receptor was considered to possibly mediate the immobility produced by inhaled anesthetics such as isoflurane and nitrous oxide. However, new evidence suggests that the role of this receptor in abolition of the movement response may be less important than previously thought. To provide further evidence supporting or challenging this view, we examined the anesthetic potencies of isoflurane and nitrous oxide in genetically modified animals with established NMDA receptor dysfunction caused by GluR&egr;1 subunit knockout. METHODS: The immobilizing properties of inhaled anesthetics in mice quantitated by the minimum alveolar anesthetic concentration (MAC) were evaluated using the classic tail clamp method. RESULTS: Compared with wild-type controls, NMDA receptor GluR&egr;1 subunit knockout mice displayed larger isoflurane MAC values indicating a resistance to the immobilizing action of isoflurane. Knockout mice were previously shown to have enhanced monoaminergic tone as a result of genetic manipulation, and this increase in MAC could be abolished in our experiments by pretreatment with the serotonin 5-hydroxytryptamine type 2A receptor antagonist ketanserin or with the dopamine D2 receptor antagonist droperidol at doses that did not affect MAC values in wild-type animals. Mutant mice also displayed resistance to the isoflurane MAC-sparing effect of nitrous oxide, but this resistance was similarly abolished by ketanserin and droperidol. Thus, resistance to the immobilizing action of inhaled anesthetics in knockout mice seems to be secondary to increased monoaminergic activation after knockout rather than a direct result of impaired NMDA receptor function. CONCLUSIONS: Our results confirm recent findings indicating no critical contribution of NMDA receptors to the immobility induced by isoflurane and nitrous oxide. In addition, they demonstrate the ability of changes secondary to genetic manipulation to affect the results obtained in global knockout studies.