Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine’s antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2 and WT showed similar contractile responses. The Atp1a2 MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca change was smaller than in WT. The enhanced constriction of Atp1a2 MCA was associated with increased cSrc activation, stronger sensitization to [Ca] and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2 mice had reduced resting blood flow through MCA in comparison with WT mice . FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

Telinius N, Kim S, Pilegaard H, Pahle E, Nielsen J, Hjortdal V, Aalkjaer C, Boedtkjer DB. The contribution of K ϩ channels to human thoracic duct contractility. Am J Physiol Heart Circ Physiol 307: H33-H43, 2014. First published April 28, 2014 doi:10.1152/ajpheart.00921.2013In smooth muscle cells, K ϩ permeability is high, and this highly influences the resting membrane potential. Lymph propulsion is dependent on phasic contractions generated by smooth muscle cells of lymphatic vessels, and it is likely that K ϩ channels play a critical role in regulating contractility in this tissue. The aim of this study was to investigate the contribution of distinct K ϩ channels to human lymphatic vessel contractility. Thoracic ducts were harvested from 43 patients and mounted in a wire myograph for isometric force measurements or membrane potential recordings with an intracellular microelectrode. Using K ϩ channel blockers and activators, we demonstrate a functional contribution to human lymphatic vessel contractility from all the major classes of K ϩ channels [ATP-sensitive K ϩ (KATP), Ca 2ϩ -activated K ϩ , inward rectifier K ϩ , and voltage-dependent K ϩ channels], and this was confirmed at the mRNA level. Contraction amplitude, frequency, and baseline tension were altered depending on which channel was blocked or activated. Microelectrode impalements of lymphatic vessels determined an average resting membrane potential of Ϫ43.1 Ϯ 3.7 mV. We observed that membrane potential changes of Ͻ5 mV could have large functional effects with contraction frequencies increasing threefold. In general, KATP channels appeared to be constitutively open since incubation with glibenclamide increased contraction frequency in spontaneously active vessels and depolarized and initiated contractions in previously quiescent vessels. The largest change in membrane voltage was observed with the KATP opener pinacidil, which caused 24 Ϯ 3 mV hyperpolarization. We conclude that K ϩ channels are important modulators of human lymphatic contractility. lymphatic vessels; membrane potential; potassium channels; thoracic duct; human LYMPHATIC COLLECTING VESSELS have an intrinsic capacity to generate phasic contractions that enable lymph to be pumped away from tissue and eventually back to the venous circulation. The intrinsic contractile activity of lymphatic vessels is triggered by an underlying electrical activity in lymphatic smooth muscle cells (LSMCs) of the vessel wall. The electrical activity of LSMCs has been measured by various techniques in several animal species but has not yet been demonstrated in human lymphatic vessels. Animal LSMCs produce spontaneous depolarization and action potentials consisting of a rapid depolarization and repolarization of the LSMC membrane potential (V m ) without a plateau phase (30). Intracellular microelectrode impalements of bovine and guinea pig mesenteric collecting lymphatic vessels (70 -500 m in diameter) have shown that the resting membrane potential (RMP) of LSMCs averages around Ϫ60 mV (27-29, 32, 33), whe...

Key pointsr We studied the effects of antihypertensive calcium channel blockers on Ca V 1.2, the predominantly expressed L-type calcium channel in the largest human lymphatic vessel, the thoracic duct.r Human lymphatic collecting vessels, both large and small, are highly-sensitive in vitro to calcium channel blockers; exposure to these drugs inhibits endogenous lymphatic contractile activity and action potentials and diminishes noradrenaline-induced phasic contractions.r In vivo administration of calcium channel blocker nifedipine to healthy volunteers did not reduce lymphatic contractile activity despite all subjects achieving nifedipine plasma concentrations comparable with those observed to affect contractile function in vitro.r These results indicate that calcium channel blocker-related oedema is unlikely to be exacerbated by an off-target effect of the drugs diminishing lymphatic pumping and fluid removal.Abstract Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l −1 nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the Ca V 1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.