Lumateperone-mediated effects on prefrontal glutamatergic receptor-mediated neurotransmission: A dopamine D1 receptor dependent mechanism.

Lumateperone is a novel drug approved for the treatment of schizophrenia in adults and depressive episodes associated with bipolar depression in adults, as monotherapy and as adjunctive therapy with lithium or valproate treatment in the United States. Lumateperone simultaneously modulates key neurotransmitters, such as serotonin, dopamine, and glutamate, implicated in serious mental illness. In patients with schizophrenia, lumateperone was shown to improve positive symptoms along with negative and depressive symptoms, while also enhancing prosocial behavior. Moreover, in patients with bipolar I or II disorder, lumateperone improved depressive symptoms as well. To further understand the mechanisms related to lumateperone's clinical response, the aim of this study was to investigate the effect of lumateperone on dopaminergic- and glutamatergic signaling in the rat medial prefrontal cortex (mPFC). We used the conditioned avoidance response (CAR) test to determine the antipsychotic-like effect of lumateperone, electrophysiology in vitro to study lumateperone's effects on NMDA- and AMPA-induced currents in the mPFC, and the neurochemical techniques microdialysis and amperometry to measure dopamine- and glutamate release in the rat mPFC. Our results demonstrate that lumateperone; i) significantly suppressed CAR in rats, indicating an antipsychotic-like effect, ii) facilitated NMDA and AMPA receptor-mediated currents in the mPFC, in a dopamine D1-dependent manner, and iii) significantly increased dopamine and glutamate release in the rat mPFC. To the extent that these findings can be translated to humans, the ability of lumateperone to activate these pathways may contribute to its demonstrated effectiveness in safely improving symptoms related to neuropsychiatric disorder including mood alterations.

Patch-clamp detection of neurotransmitters in capillary electrophoresis.

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Olanzapine and clozapine but not haloperidol reverse subchronic phencyclidine-induced functional hyperactivity of N-methyl-D-aspartate receptors in pyramidal cells of the rat medial prefrontal cortex.

In the present study, we have investigated and compared the ability of olanzapine, clozapine and haloperidol to modulate phencyclidine (PCP)-induced effect in pyramidal cells of the medial prefrontal cortex (mPFC) of rats using the techniques of intracellular recording and voltage-clamp. Subchronic treatment of rats with PCP (2 mg/kg, b.i.d., 7 days, 48-60 h withdrawal) produced: (1) a depolarized resting membrane potential, a decrease of slow after hyperpolarization (sAHP) and spike frequency adaptation, (2) a shift of the concentration response curve of N-methyl-D-aspartate (NMDA), but not (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), to the left, (3) a decrease of the paired pulse facilitation (PPF) with an increase of excitatory postsynaptic current variance (EPSC variance), and (4) a reduction of the blockade of NMDA response by in vitro application of PCP. Repeated treatment with either olanzapine or clozapine, but not haloperidol, completely prevented the aforementioned subchronic PCP-induced effects. The present results indicate that the atypical antipsychotic drugs (APDs) clozapine and olanzapine share a common property in preventing subchronic PCP-induced functional hyperactivity of NMDA receptors.

Subchronic treatment with either clozapine, olanzapine or haloperidol produces a hyposensitive response of the rat cortical cells to N-methyl-D-aspartate.

Using the technique of intracellular recording in in vitro brain slice preparations, we examined the effects produced by repeated administration of the antipsychotic drugs clozapine, olanzapine and haloperidol, on N-methyl-D-aspartic acid-induced responses in pyramidal cells of the rat medial prefrontal cortex. Rats were anesthetized and decapitated 24h after the conclusion of daily intraperitoneal injection with either clozapine (25mg/kg), olanzapine (1, 5 or 10mg/kg) or haloperidol (0.5mg/kg) for 21 days, and the slices from medial prefrontal cortex were used for electrophysiological recordings. The concentration-response curves for N-methyl-D-aspartic acid to activate cortical cells shifted markedly to the right in rats which received the subchronic antipsychotic drug treatment, compared with those obtained from rats which received repeated injections of vehicle (1ml/kg/day, i.p. for 21 days). In addition, repeated exposure to antipsychotic drugs caused a significant reduction in the ability of these antipsychotic drugs to augment the N-methyl-D-aspartic acid-induced inward current in pyramidal cells of the rat medial prefrontal cortex. Repeated administration of haloperidol, but not clozapine or olanzapine, significantly hyperpolarized the resting membrane potential and increased membrane resistance in pyramidal cells of the medial prefrontal cortex. Moreover, subchronic treatment with haloperidol, but not clozapine or olanzapine, depressed (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-induced responses. The desensitized response of medial prefrontal cortex cells to N-methyl-D-aspartic acid could be the result of a compensatory response to the facilitating action of antipsychotic drugs on N-methyl-D-aspartic acid receptor-mediated transmission. The inhibitory action of haloperidol on (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid responses may also contribute to the rightward shift of the N-methyl-D-aspartic acid concentration-response curve.Thus, the present study suggests that the atypical antipsychotic drugs, clozapine and olanzapine, as well as the typical antipsychotic drug haloperidol strongly modulate glutamatergic transmission after prolonged treatment. This might be an important factor in the mechanisms by which these drugs alleviate symptoms in schizophrenic patients.

Protein kinase C is involved in clozapine's facilitation of N-methyl-D-aspartate- and electrically evoked responses in pyramidal cells of the medial prefrontal cortex.

We have previously shown that the atypical antipsychotic drug clozapine facilitates N-methyl-D-aspartate (NMDA)- and electrically evoked responses in pyramidal cells of the medial prefrontal cortex (mPFC). In the present study, we investigated the role of protein kinase C (PKC) in the action of clozapine. Bath administration of the PKC activator phorbol-12-myristate 13-acetate (PMA), but not the inactive isomer 4alpha-PMA, significantly enhanced the NMDA-evoked inward current and electrically evoked excitatory postsynaptic currents. Chelerythrine, a selective blocker of PKC, completely prevented the potentiating action produced by either clozapine or PMA on these currents in the mPFC cells. Intracellular injection of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. Of the PKC inhibitor PKC-I, but not the control substance PKC-S, through the recording electrode totally blocked clozapine's potentiating effect, indicating that a post-synaptic expressed PKC is critically involved in the augmenting action of clozapine on NMDA-evoked currents. To further test the role of PKC in mediating the augmenting action of clozapine, we performed experiments in PKCgamma mutant and wild-type mice. In contrast to results in pyramidal cells from rats or wild-type mice, neither clozapine nor PMA was able to potentiate NMDA-induced currents in the mPFC from the PKCgamma mutant mice. Taken together, these results suggest that the PKC signal transduction pathway is critically involved in the facilitating action of clozapine on the NMDA-induced responses in pyramidal cells of the mPFC.

Nitric oxide is involved in nicotine-induced burst firing of rat ventral tegmental area dopamine neurons.

In the present study, using single cell recordings in vivo and intracellular recordings in vitro from midbrain slices, the role of N-methyl-d-aspartate (NMDA) receptor signaling on firing activity in ventral tegmental area dopamine neurons elicited by nicotine was investigated in the rat. In accordance with previous studies, systemic nicotine (0.5 mg/kg s.c.) increased both firing rate and burst firing of dopamine neurons in vivo, and bath-applied nicotine (10 microM) increased firing rate in vitro. The competitive NMDA receptor antagonist CGP39551 (2.5 mg/kg i.p.) inhibited nicotine's effects on burst firing and also attenuated the nicotine-induced increase in firing rate. Moreover, although the nitric oxide (NO)-synthase inhibitor N-nitro-l-arginine-methyl-ester (l-NAME; 5.0 mg/kg i.p.) had no effect on cell firing by itself, it prevented the response to nicotine in vivo. In contrast, l-NAME (100 microM) did not influence nicotine's effect on dopamine cell firing in vitro, suggesting that the effect of l-NAME seen in vivo is dependent on presynaptic afferent input. The present study confirms previous results suggesting that the effect of systemically administered nicotine is in part presynaptic and mediated via NMDA receptors. The data also indicate that NO plays an important role in the previously demonstrated, indirect, glutamate-mediated excitation of these neurons by nicotine. By inference, our results provide additional support for the involvement of NO in nicotine dependence.

Activation of beta-adrenoceptors opens calcium-activated potassium channels in astroglial cells.

In the present study, effects of the alpha(2)- and beta-adrenoceptor agonists clonidine and isoproterenol on astrocytes in astroglial/neuronal cocultures from rat cerebral cortex were evaluated. The calcium- and potassium-sensitive dyes fura-2 and potassium-binding benzofuran isophtalate (PBFI) were used to study alterations in intracellular concentrations of calcium ([Ca(2+)](i)) and potassium ([K(+)](i)), respectively, while the perforated patch clamp technique was used to analyze transmembrane currents. Exposure to isoproterenol or clonidine elicited an immediate increase in [Ca(2+)](i) that was totally abolished in calcium-free extracellular media. Isoproterenol also decreased [K(+)](i), but clonidine did not. The reduction in [K(+)](i) was inhibited in Ca(2+)-free media. As evaluated with the perforated patch technique, isoproterenol (10(-6)-10(-4) M) induced a slowly developing and long lasting outward current that also was totally abolished in calcium-free buffer. This current was blocked by external tetraethylammonium (TEA, 10 mM) and charybdotoxin (ChTX, 10 nM), but was not affected by apamin (50 nM). The current-to-voltage (I-V) relationships for the isoproterenol-induced currents showed a markedly negative reversal potential, -96 mV+/-7, (mean+/-S.D., n=5). These results suggest that the stimulation of astroglial beta-adrenoceptors by isoproterenol opens calcium-activated potassium channels (K((Ca))). Preincubation with forskolin significantly increased the isoproterenol-induced currents compared with controls, indicating that the opening of astroglial K((Ca)) channels after beta-adrenergic stimulation not only depends on [Ca(2+)](i) but also synergistically involves the cAMP transduction system to which beta-adrenoceptors are known to be positively coupled.

Bicarbonate-sensitive cysteine induced elevation of extracellular aspartate and glutamate in rat hippocampus in vitro.

The effect of different concentrations of cysteine (0.125, 0.25, 0.5 and 1 mM) on the net efflux of endogenous amino acids was studied by the incubation of rat hippocampal slices. Addition of cysteine (1 mM) in bicarbonate containing low K+ medium (5 min) selectively increased the basal net efflux of glutamate and aspartate by 370% and 396%, respectively. High K+ media (50 mM) containing cysteine (1 mM) evoked the net efflux of glutamate and aspartate by 1454% and 1019%, respectively. The corresponding effects in control slices without cysteine were 669% and 404%, respectively. No changes were observed on the concentrations of GABA, glutamine and taurine. The cysteine oxidation products, cysteine sulfinate (0.5 microM) and cystine (0.25 mM) were without effects. The effect of cysteine (0.5 mM) was dramatically reduced in media with no added bicarbonate/CO2. Thus, cysteine in a bicarbonate-sensitive manner selectively increases the extracellular concentration of excitotoxic amino acids in adult rat brain in vitro, possibly by interfering with the carrier-mediated glutamate uptake/release.

Expression of Ca(2+)-ion permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in Xenopus oocytes injected with total RNA from human epileptic temporal lobe.

By using the Xenopus oocyte as an expression system, we have performed a series of experiments in order to examine the divalent cation-permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors from the human epileptic temporal lobe. Xenopus oocytes, injected with total RNA from the epileptic temporal lobe, were tested for expression of receptors by a conventional two electrode voltage-clamp technique. Administration of glutamate and AMPA gave small or no clear current responses, whereas kainate produced large inward non-desensitizing currents. The current responses evoked by kainate were concentration dependent. Experimental data gave a Hill coefficient of 1.06 and an EC50 value of 87 microM. The current to voltage relationship showed an inward rectification and when the concentration of divalent cations were enhanced, there was a shift in the reversal potential from -11 mV (2 mM Ca2+) to 12 mV (60 mM Ba2+). This yielded a pBa2+/pK+ permeability ratio of 1.6 when the constant field equation was used. The amplitude of the currents evoked by 600 microM kainate in solutions containing higher Ba(2+)-ion concentrations was markedly diminished (46% in 10 mM Ba(2+)- and 75% in 60 mM Ba(2+)-solution), when compared to those obtained in normal Ringer's solution, suggesting interactions between different cation species and/or screening of surface charges.

Ca2+ ion permeability properties of (R,S) alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in isolated interneurons from the olfactory bulb of the rat.

The aim of the study was to investigate the divalent cation permeability of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors expressed in interneurons of the olfactory bulb. Kainic acid (KA) was used as agonist to activate AMPA-receptor-mediated currents, which were recorded with the use of the patch-clamp technique. In interneurons acutely isolated from the olfactory bulb, the current responses to KA showed linear/outwardly rectifying current-voltage (I-V) relationships with a positive average reversal potential of +7 mV in normal external medium (1 mM Ca2+, 1 mM Mg2+). Raising the external Ca2+ concentration to 10 mM suppressed the amplitude, whereas omission of Ca2+ enhanced the amplitude of the current. Spectral analysis of the increase in current variance produced by KA indicated that the decreased amplitude observed in 10 mM Ca2+ was accompanied by a reduction in the apparent single-channel conductance. Raising the concentration of Mg2+ from 1 to 10 mM had a weak depressant effect on the KA-evoked current amplitude. No shift in the reversal potential was observed when the concentration of Ca2+ or Mg2+ was changed from 1 to 10 mM. Increasing the external medium concentration of Ca2+ to 60 mM not only further depressed the amplitudes of the KA-evoked currents but also gave a pronounced leftward shift in the average reversal potential to -32 +/- 9 (SE) mV (N = 7). For neurons in primary culture, current responses to KA also showed linear/outwardly rectifying I-V relationships with a positive average reversal potential in normal external medium. Substituting N-methylglucamine for Na+ and increasing the Ca2+ concentration to 10 mM gave a leftward shift in the average reversal potential from +9 +/- 3 mV to -47 +/- 4 mV (N = 11) and caused a marked reduction in the amplitude of the KA-evoked currents at negative potentials. The permeability properties of the studied AMPA receptors were well predicted by the Eyring rate model (symmetrical, 2 barriers, 1 site). The model gave a pCa2+/pK+ permeability ratio of 0.06 for acutely isolated interneurons and 0.14 for interneurons in primary culture. The constant field theory, which failed to successfully reproduce all the experimental data, gave corresponding low permeability ratios of 0.18 and 0.40 for acutely isolated cells and cells in primary culture, respectively. Thus it is concluded that interneurons in the olfactory bulb mainly express AMPA receptors with low permeability to Ca2+ ions.

Patch clamp detection in capillary electrophoresis.

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

Biphasic modulation of NMDA-induced responses in pyramidal cells of the medial prefrontal cortex by Y-931, a potential atypical antipsychotic drug.

Similar to the effects produced by the atypical antipsychotic drugs (APDs) clozapine and olanzapine, Y-931 [8-fluoro-12-(4-methylpiperazin-1-yl)-6H-[1]benzothieno[2,3-b][1,5]benzodiazepine maleate, a purported atypical APD] effectively facilitated N-methyl-D-aspartate (NMDA)-induced, but not (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-evoked, responses in pyramidal cells of the rat medial prefrontal cortex (mPFC). Similar to olanzapine and clozapine, the concentration-response curve of Y-931 in these experiments was biphasic. At present, the mechanisms behind the biphasic modulatory actions of Y-931 and olanzapine on NMDA-induced currents in the mPFC are not clear. In addition to augmenting NMDA responses, Y-931 prevented the phencyclidine (PCP)-induced block of the NMDA responses and increased the amplitudes and durations of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of the forceps minor. Overall, our findings suggest that APDs, particularly the atypical ones, share a common property in that they facilitate NMDA receptor-mediated transmission in the mPFC and perhaps other functionally related limbic structures as well, which could be the cellular basis for their ability to alleviate some schizophrenic negative symptoms and cognitive dysfunctions.

Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells.

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.

Characterization of single-cell electroporation by using patch-clamp and fluorescence microscopy.

Electroporation of single NG108-15 cells with carbon-fiber microelectrodes was characterized by patch-clamp recordings and fluorescence microscopy. To minimize adverse capacitive charging effects, the patch-clamp pipette was sealed on the cell at a 90(o) angle with respect to the microelectrodes where the applied potential reaches a minimum. From transmembrane current responses, we determined the electric field strengths necessary for ion-permeable pore formation and investigated the kinetics of pore opening and closing as well as pore open times. From both patch-clamp and fluorescence microscopy experiments, the threshold transmembrane potentials for dielectric breakdown of NG108-15 cells, using 1-ms rectangular waveform pulses, was approximately 250 mV. The electroporation pulse preceded pore formation, and analyte entry into the cells was dictated by concentration, and membrane resting potential driving forces. By stepwise moving a cell out of the focused field while measuring the transmembrane current response during a supramaximal pulse, we show that cells at a distance of approximately 30 microm from the focused field were not permeabilized.