Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions.

(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

Intrahippocampal injection of a selective blocker of NMDA receptors containing the GluN2B subunit, Ro25-6981, increases glutamate neurotransmission and induces antidepressant-like effects.

Major depressive disorder (MDD) is a serious public health problem, as it is the most common psychiatric disorder worldwide. Antidepressant drugs increase adult hippocampal neurogenesis, which is required to induce some behavioral effects of antidepressants. Adult-born granule cells in the dentate gyrus (DG) and the glutamate receptors subunits 2 (GluN2B) subunit of N-methyl-D-aspartate (NMDA) ionotropic receptors play an important role in these effects. However, the precise neurochemical role of the GluN2B subunit of the NMDA receptor on adult-born GCs for antidepressant-like effects has yet to be elucidated. The present study aims to explore the contribution of the GluN2B-containing NMDA receptors in the ventral dentate gyrus (vDG) to the antidepressant drug treatment using a pharmacological approach. Thus, (αR)-(4-hydroxyphenyl)-(ßS)-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro25-6981), a selective antagonist of the GluN2B subunit, was acutely administered locally into the ventral DG (vDG, 1 µg each side) following a chronic fluoxetine (18 mg/kg/day) treatment-known to increase adult hippocampal neurogenesis-in a mouse model of anxiety/depression. Responses in a neurogenesis-dependent task, the novelty suppressed feeding (NSF), and neurochemical consequences on extracellular glutamate and gamma-aminobutyric acid (GABA) levels in the vDG were measured. Here, we show a rapid-acting antidepressant-like effect of local Ro25-6981 administration in the NSF independent of fluoxetine treatment. Furthermore, we revealed a fluoxetine-independent increase in the glutamatergic transmission in the vDG. Our results suggest behavioral and neurochemical effects of GluN2B subunit independent of serotonin reuptake inhibition.

Intranasal (R, S)-ketamine delivery induces sustained antidepressant effects associated with changes in cortical balance of excitatory/inhibitory synaptic activity.

In 2019, an intranasal (IN) spray of esketamine SPRAVATO® was approved as a fast-acting antidepressant by drug Agencies US FDA and European EMA. At sub-anesthetic doses, (±)-ketamine, a non-competitive glutamate N-methyl-d-aspartate (NMDA) receptor antagonist, increases the overall excitability of the medial prefrontal cortex (mPFC), an effect being essential for its rapid antidepressant activity. We wondered if this effect of ketamine could come from changes in the balance between neuronal excitation and inhibition (E/I balance) in the mPFC. Here, we performed a preclinical approach to study neurochemical and behavioral responses to a single IN ketamine dose in BALB/cJ mice, a strain more sensitive to stress. By using in vivo microdialysis, we measured cortical E/I balance as the ratio between glutamate to GABA extracellular levels 24 h post-ketamine. We found, for the first time, that E/I balance was shifted in favor of excitation rather than inhibition in the mPFC but more robustly with IN KET than with a single intraperitoneal (IP) dose. Increases in plasma and brain ketamine, norketamine and HNKs levels suggest different metabolic profiles of IP and IN ketamine 30 min post-dose. A significantly larger proportion of ketamine and HNKs in the brain are derived from the IN route 30 min post-dose. It may be linked to the greater magnitude in E/I ratio following IN delivery relative to IP at t24 h. This study suggests that both IP and IN are effective brain delivery methods inducing similar sustained antidepressant efficacy of KET, but the way they induced neurotransmitter changes is slightly different.

Cortical and raphe GABAA, AMPA receptors and glial GLT-1 glutamate transporter contribute to the sustained antidepressant activity of ketamine.

At sub-anaesthetic doses, ketamine, a non competitive N-methyl-d-aspartate (NMDA) receptor antagonist, has demonstrated remarkable and rapid antidepressant (AD) efficacy in patients with treatment-resistant depression (TRD). However, its mechanism of action of ketamine is not fully understood. Since comorbid depression and anxiety disorders often occur, GABAergic/inhibitory and glutamatergic/excitatory drug treatments may be co-administered in these patients. Information regarding this combination is critical to establish efficacy or treatment restrictions to maximize translation from animal models to TRD patients, effectiveness and safety. To assess the specific role of excitatory/inhibitory neurotransmission in the medial prefrontal cortex-raphe nuclei (mPFC-DRN) circuit in the sustained antidepressant-like activity (AD) of ketamine (at t24h post dose), AMPA-R antagonist (intra-DRN) and GABAA-R agonist (intra-mPFC) were co-administered with ketamine (intra-mPFC). Twenty-four hours later, responses in the forced swim test (FST) and neurochemical consequences on extracellular mPFC glutamate, GABA and 5-HT levels were measured in BALB/cJ mice. Intra-DRN NBQX prevented the sustained AD-like activity of ketamine evidenced by decreases in FST swimming duration and blunted cortical 5-HText and Gluext. Intra-mPFC muscimol blocked ketamine AD-like activity and its effects on cortical 5-HText. Moreover, a selective glutamate transporter GLT-1 inhibitor, dihydrokainic acid (DHK) locally perfused into the mPFC produced an AD-like activity at t24h associated with robust increases in mPFC 5-HText, Gluext and GABAext. Thus, the sustained AD-like activity of ketamine is triggered by AMPA-R activation in the DRN and 5-HT - glutamate release in the mPFC, but limited by GABAA-R activation - GABA release in the mPFC. The local blockade of GLT-1 in the mPFC also mimics the rapid responses of ketamine, thus highlighting the role of neuronal-glial adaptation in these effects. These results also suggests the need to test for the concomitant prescription of ketamine and BZD to see whether its sustained antidepressant activity is maintained in TRD patients.

Rapid analysis of glutamate, glutamine and GABA in mice frontal cortex microdialysis samples using HPLC coupled to electrospray tandem mass spectrometry.

In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and l-glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC-MS/MS quantification of l-glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core-shell ammonium-sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25 mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of l-glutamic acid, glutamine and GABA, as well as the internal standards [d6]-GABA and [d5]-glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63 ng/ml for GABA, 1.25 ng/ml for l-glutamic acid and 3.15 ng/ml for glutamine, and the intra-day and inter-day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium-evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.

Firing and intrinsic properties of antennal lobe neurons in the Noctuid moth Agrotis ipsilon.

The antennal lobe (AL) of the Noctuid moth Agrotis ipsilon has emerged as an excellent model for studying olfactory processing and its plasticity in the central nervous system. Odor-evoked responses of AL neurons and input-to-output transformations involved in pheromone processing are well characterized in this species. However, the intrinsic electrical properties responsible of the firing of AL neurons are poorly known. To this end, patch-clamp recordings in current- and voltage-clamp mode from neurons located in the two main clusters of cell bodies in the ALs were combined with intracellular staining on A. ipsilon males. Staining indicated that the lateral cluster (LC) is composed of 85% of local neurons (LNs) and 15% of projection neurons (PNs). The medial cluster (MC) contains only PNs. Action potentials were readily recorded from the soma in LNs and PNs located in the LC but not from PNs in the MC where recordings showed small or no action potentials. In the LC, the spontaneous activity of about 20% of the LNs presented irregular bursts while being more regular in PNs. We also identified a small population of LNs lacking voltage-gated Na(+) currents and generating spikelets. We focused on the firing properties of LNs since in about 60% of LNs, but not in PNs, action potentials were followed by depolarizing afterpotentials (DAPs). These DAPs could generate a second action potential, so that the activity was composed of action potential doublets. DAPs depended on voltage, Ca(2+)-channels and possibly on Ca(2+)-activated non-specific cationic channels. During steady state current injection, DAPs occurred after each action potential and did not require high-frequency firing. The amplitude of DAPs increased when the interspike interval was small, typically within bursts, likely arising from a Ca(2+) build up. DAPs were more often found in bursting than in non-bursting LNs but do not support bursting activity. DAPs and spike doublets also occurred during odor-evoked activity suggesting that they can mediate olfactory integration in the AL.

Anopheles gambiae mosquito isolated neurons: a new biological model for optimizing insecticide/repellent efficacy.

To understand better the mode of action of insecticides and repellents used in vector-borne diseases control, we developed a new biological model based on mosquito neurons isolated from adults Anopheles gambiae heads. This cellular model is well adapted to multidisciplinary approaches: electrophysiology, pharmacology, molecular biology and biochemical assays. Using RT-PCR, we demonstrated that isolated neurons express the nicotinic acetylcholine receptor subunit α1 (Agα1 nAchR), two acetylcholinesterases (AChE-1 and AChE-2) and three voltage-gated ion channels required for membrane excitability (AgCav1, AgNav1 and AgKv1). In order to correlate the expression of the different transcripts, encoding functional AgNav channel, nAChR receptor and AChE enzymes detected by RT-PCR, with electrophysiological activity we used patch-clamp technique. We revealed that AgNav and AChE which are targeted by insecticide and/or repellent were sensitive to the pyrethroid permethrin and to the repellent DEET, respectively. In addition, using colorimetric method, we also showed that AChE was sensitive to the carbamate propoxur. These results indicated that this novel neuronal mosquito model will lead to molecular and functional characterization of insecticide/repellent targets and appears as a powerful tool to investigate the development of highly specific and effective strategies for disease vector control.

How does calcium-dependent intracellular regulation of voltage-dependent sodium current increase the sensitivity to the oxadiazine insecticide indoxacarb metabolite decarbomethoxylated JW062 (DCJW) in insect pacemaker neurons?

Decarbomethoxylated JW062 (DCJW), the active component of the oxadiazine insecticide (S)-methyl 7-chloro-2,5-dihydro-2-[[(methoxycarbonyl)[4-(trifluoromethoxy)phenyl] amino]carbonyl] indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-carboxylate (DPX-JW062) (indoxacarb), was tested on 2 inward voltage-dependent sodium currents (named INa1 and INa2) expressed in short-term cultured dorsal unpaired median neurons of the cockroach Periplaneta americana. Under whole-cell voltage-clamp conditions, application of DCJW resulted in a biphasic dose-dependent inhibition of the global sodium current amplitude illustrating the differing sensitivity of sodium channels to DCJW. INa2 was less sensitive to DCJW [half-maximal inhibitory concentration (IC(50)) = 1.6 microM] compared with INa1 (IC(50) = 1.7 nM). Although a previous study demonstrated that INa1 was regulated by the cAMP/protein kinase A cascade, we showed that INa2 was mainly regulated in an opposite way by the activation of calcium-calmodulin-dependent protein phosphatase 2B (PP2B) and calcium-calmodulin-dependent protein kinase II (CaM-kinase II). Furthermore, we demonstrated that activation of CaM-kinase II by intracellular calcium via the calcium-calmodulin complex affected the sensitivity of INa2 channels to DCJW. By increasing the intracellular calcium concentration and/or using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (a calcium chelator), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) (a calmodulin inhibitor), cyclosporine A (a PP2B inhibitor), and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) (a CaM-kinase II inhibitor), we revealed that activation of CaM-kinase II was involved in the modulation of the voltage dependence of steady-state inactivation and that the CaM-kinase II pathway activated by elevation of the intracellular calcium concentration might render INa2 channels approximately 3000-fold more sensitive to DCJW. These results indicated that manipulating specific intracellular signaling pathways involved in the regulation of sodium channels might have fundamental consequences for the sensitivity of insects to insecticides. This finding reveals an exciting research area that could lead to improvement in the efficiency of insecticides.

Differential regulation of two distinct voltage-dependent sodium currents by group III metabotropic glutamate receptor activation in insect pacemaker neurons.

Using whole cell patch-clamp technique and immunocytochemistry on adult dorsal unpaired median (DUM) neurons isolated from the cockroach Periplaneta americana CNS, we reported the characterization of a native mGluR, sharing pharmacological properties with vertebrate metabotropic glutamate receptor III (mGluRIII) that regulated voltage-dependent sodium current (I(Na)). The global I(Na) was dissociated by means of l-glutamate sensitivity, deactivation time constant, voltage dependence of activation and inactivation, recovery from inactivation, and intracellular regulation process. These two currents were respectively designated I(Na1) and I(Na2) for l-glutamate-sensitive and -insensitive sodium currents. l-glutamate selectively reduced I(Na1) by an increase of intracellular cAMP level. Using different activators and/or inhibitors of G proteins and cAMP/PKA cascade, together with St-Ht31 (an inhibitor of PKA binding to AKAP) and AKAP-79 antibodies, we established that mGluRIII was linked to I(Na1) by a Gi/o and a suspected Gs protein. According to the activated signaling pathway, l-glutamate elevated the cAMP level, which thereby activated cytosolic PKA and released PKA bound to AKAP. As expected from both biophysical and pharmacological studies, we showed that, through an inhibition of I(Na1), l-glutamate increased DUM neuron spontaneous electrical activity. These results indicated that such mGluRIII-activated dual processes provided a new physiological control of pacemaker neuronal firing.