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 γ-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.
Abstract Muscle fiber force production is determined by the excitation frequency of motor nerves, which induce transient increases in cytoplasmic free Ca 2+ concentration ([Ca 2+ ] i ) and the force-generating capacity of the actomyosin cross-bridges. Previous studies suggest that, in addition to altered cross-bridge properties, force changes during dynamic (concentric or eccentric) contraction might be affected by Ca 2+ -dependent components. Here we investigated this by measuring [Ca 2+ ] i and force in mouse muscle fibers undergoing isometric, concentric, and eccentric contractions. Intact single muscle fibers were dissected from the flexor digitorum brevis muscle of mice. Fibers were electrically activated isometrically at 30–100 Hz and after reaching the isometric force plateau, they were actively shortened or stretched. We calculated the ratio (relative changes) in force and [Ca 2+ ] i attained in submaximal (30 Hz) and near-maximal (100 Hz) contractions under isometric or dynamic conditions. Tetanic [Ca 2+ ] i was similar during isometric, concentric and eccentric phases of contraction at given stimulation frequencies while the forces were clearly different depending on the contraction types. The 30/100 Hz force ratio was significantly lower in the concentric (44.1 ± 20.3%) than in the isometric (50.3 ± 20.4%) condition ( p = 0.005), whereas this ratio did not differ between eccentric and isometric conditions ( p = 0.186). We conclude that the larger force decrease by decreasing the stimulation frequency during concentric than during isometric contraction is caused by decreased myofibrillar Ca 2+ sensitivity, not by the decreased [Ca 2+ ] i .
Asenapine is a novel psychopharmacologic agent being developed for schizophrenia and bipolar disorder. Like clozapine, asenapine facilitates cortical dopaminergic and N-methyl-D-aspartate (NMDA) receptor-mediated transmission in rats. The facilitation of NMDA-induced currents in cortical pyramidal cells by clozapine is dependent on dopamine and D(1) receptor activation. Moreover, previous results show that clozapine prevents and reverses the blockade of NMDA-induced currents and firing activity in the pyramidal cells by the noncompetitive NMDA receptor antagonist phencyclidine (PCP). Here, we investigated the effects of asenapine in these regards using intracellular electrophysiological recording in vitro. Asenapine (5 nM) significantly facilitated NMDA-induced currents (162 ± 15% of control) in pyramidal cells of the medial prefrontal cortex (mPFC). The asenapine-induced facilitation was blocked by the D(1) receptor antagonist SCH23390 (1 μM). Furthermore, the PCP-induced blockade of cortical NMDA-induced currents was effectively reversed by 5 nM asenapine. Our results demonstrate a clozapine-like facilitation of cortical NMDA-induced currents by asenapine that involves prefrontal dopamine and activation of D(1) receptors. Asenapine and clozapine also share the ability to reverse functional PCP-induced hypoactivity of cortical NMDA receptors. The ability of asenapine to increase both cortical dopaminergic and NMDA receptor-mediated glutamatergic transmission suggests that this drug may have an advantageous effect not only on positive symptoms in patients with schizophrenia, but also on negative and cognitive symptoms.
Escitalopram, the S-enantiomer of citalopram, possesses superior efficacy compared to other selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depression. Escitalopram binds to an allosteric site on the serotonin transporter, which further enhances the blockade of serotonin reuptake, whereas R-citalopram antagonizes this positive allosteric modulation. Escitalopram's effects on neurotransmitters other than serotonin, for example, dopamine and glutamate, are not well studied. Therefore, we here studied the effects of escitalopram, citalopram, and R-citalopram on dopamine cell firing in the ventral tegmental area, using single-cell recording in vivo and on NMDA receptor-mediated currents in pyramidal neurons in the medial prefrontal cortex using in vitro electrophysiology in rats. The cognitive effects of escitalopram and citalopram were also compared using the novel object recognition test. Escitalopram (40-640 μg/kg i.v.) increased both firing rate and burst firing of dopaminergic neurons, whereas citalopram (80-1280 μg/kg) had no effect on firing rate and only increased burst firing at high dosage. R-citalopram (40-640 μg/kg) had no significant effects. R-citalopram (320 μg/kg) antagonized the effects of escitalopram (320 μg/kg). A very low concentration of escitalopram (5 nM), but not citalopram (10 nM) or R-citalopram (5 nM), potentiated NMDA-induced currents in pyramidal neurons. Escitalopram's effect was antagonized by R-citalopram and blocked by the dopamine D(1) receptor antagonist SCH23390. Escitalopram, but not citalopram, improved recognition memory. Our data suggest that the excitatory effect of escitalopram on dopaminergic and NMDA receptor-mediated neurotransmission may have bearing on its cognitive-enhancing effect and superior efficacy compared to other SSRIs in major depression.
Preclinical data show that addition of the selective norepinephrine transporter (NET) inhibitor reboxetine to raclopride, a typical D2/3 antagonist, increases its antipsychotic efficacy and, in parallel, enhances dopamine output in the medial prefrontal cortex (mPFC), effects similar to those of clozapine. Subsequent c linical results suggest that adding reboxetine to a stable treatment with various antipsychotic drugs (APDs) may indeed improve positive, negative and depressive symptoms in schizophrenia. Here we investigated in rats the effects of concomitant administration of reboxetine and the second generation APD olanzapine on: i) antipsychotic effect using the conditioned avoidance response (CAR) test, ii) extrapyramidal side effect (EPS) liability using the catalepsy test, iii) dopamine efflux in the mPFC and the nucleus accumbens using in vivo microdialysis in freely moving animals, and iv ) cortical NMDA receptor-mediated transmission using intracellular electrophysiological recording in vitro . Reboxetine (6 mg/kg) enhanced the suppression of CAR induced by a low (1.25 mg/kg), but not a high (2.5 mg/kg) dose of olanzapine without any concomitant catalepsy . Addition of reboxetine to the low dose of olanzapine also induced a large increase in cortical dopamine outflow as well as a markedly enhanced NMDA receptor-mediated transmission, in analogy with clozapine. These data help explain how adjunctive treatment with a NET inhibitor may enhance the efficacy of olanzapine in schizophrenia, yet without increasing EPS liability, including an antidepressant action, and allow for a reduction of dosage as well as dose-related side effects such as weight gain, which has already been observed clinically.
The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.
The α2 adrenoceptor antagonist idazoxan enhances antipsychotic efficacy of classical dopamine D2 antagonists in treatment-resistant schizophrenia. The mechanisms are not fully understood, but we have previously shown that the combination of idazoxan with the D2/3 receptor antagonist raclopride, similarly to clozapine but not classical antipsychotic drugs, augments dopamine efflux in the prefrontal cortex, and also generates an enhanced suppression of the conditioned avoidance response. We have now investigated the effects of clozapine, raclopride, idazoxan and the combination of raclopride and idazoxan on (i) electrically evoked excitatory post-synaptic potentials and currents in pyramidal cells of the rat medial prefrontal cortex, using intracellular electrophysiological recording in vitro, (ii) the impaired cognitive function induced by the selective N-methyl-d-aspartate (NMDA) receptor antagonist MK-801, using the 8-arm radial maze test, (iii) the in-vivo D2, α2A and α2C receptor occupancies of these pharmacological treatments, using ex-vivo autoradiography. Whereas neither idazoxan nor raclopride alone had any effect, the combination exerted the same facilitation of glutamatergic transmission in rat prefrontal pyramidal neurons as clozapine, and this effect was found to be mediated by dopamine acting at D1 receptors. Similarly to clozapine, the combination of idazoxan and raclopride also completely reversed the working-memory impairment in rats induced by MK-801. Moreover, these effects of the two treatment regimes were obtained at similar occupancies at D2, α2A and α2C receptors respectively. Our results provide novel neurobiological and behavioural support for a pro-cognitive effect of adjunctive use of idazoxan with antipsychotic drugs that lack appreciable α2 adrenoceptor-blocking properties, and define presynaptic α2 adrenoceptors as major targets in antipsychotic drug development.
