Bartłomiej Szulczyk

D1 DOPAMINERGIC CONTROL OF G PROTEIN-DEPENDENT INWARD RECTIFIER K+ (GIRK)-LIKE CHANNEL CURRENT IN PYRAMIDAL NEURONS OF THE MEDIAL PREFRONTAL CORTEX

Pyramidal neurons of the medial prefrontal cortex (mPFC) exhibit dopamine-dependent prolonged depolarization, which may lead to persistent activity. Persistent activation of prefrontal cortex neurons has been proposed to underlie the working memory process. The purpose of our study was to test the hypothesis that activation of D(1) dopamine receptors leads to inhibition of G protein-dependent inward rectifier K(+) (GIRK) channels, thereby supporting the prolonged depolarization of mPFC pyramidal neurons. Experiments were performed on 3-week-old rats. GIRK-like channel currents recorded from pyramidal neurons showed the following properties at -75 mV: open probability (NPo), 2.5+/-0.3 x 10(-3); mean open time, 0.53+/-0.05 ms; and conductance, 29.9+/-1.6 pS (n=60). The channel currents were strongly inward-rectified. GIRK channel currents were reversibly inhibited by the D(1) agonists SKF 38393 (10 microM) and SKF 81297 (10 microM). This inhibition was abolished by prior application of a dopamine receptor antagonist and by application of the membrane-permeable protein kinase C inhibitors chelerythrine chloride (3 microM) and calphostin C (10 microM). It was also found that the application of D(1) dopamine receptor agonists or GIRK channel inhibitors evoked depolarization of mPFC pyramidal neurons in rats. Moreover, prior application of a GIRK channel blocker eliminated the depolarizing effect of D(1) agonists. We conclude that activation of D(1) dopamine receptors may lead to inhibition of GIRK channel currents that may, in turn, lead to the prolonged depolarization of mPFC pyramidal neurons in juvenile rats.

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons.

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.

Properties of BK-type Ca ++ -dependent K + channel currents in medial prefrontal cortex pyramidal neurons in rats of different ages

The medial prefrontal cortex (PFC) is involved in cognitive functions, which undergo profound changes during adolescence. This alteration of the PFC function derives from neuron activity, which, in turn, may depend on age-dependent properties and the expression of neuronal ion channels. BK-type channels are involved in controlling both the Ca++ ion concentration in the cell interior and cell excitability. The purpose of this study was to test the properties of BK currents in the medial PFC pyramidal neurons of young (18- to 22-day-old), adolescent (38- to 42-day-old), and adult (60- to 65-day-old) rats. Whole-cell currents evoked by depolarizing voltage steps were recorded from dispersed medial PFC pyramidal neurons. A selective BK channel blocker – paxilline (10 μM) – irreversibly decreased the non-inactivating K+ current in neurons that were isolated from the young and adult rats. This current was not significantly affected by paxilline in the neurons obtained from adolescent rats. The properties of single-channel K+ currents were recorded from the soma of dispersed medial PFC pyramidal neurons in the cell-attached configuration. Of the K+ channel currents that were recorded, ~90% were BK and leak channel currents. The BK-type channel currents were dependent on the Ca++ concentration and the voltage and were inhibited by paxilline. The biophysical properties of the BK channel currents did not differ among the pyramidal neurons isolated from young, adolescent, and adult rats. Among all of the recorded K+ channel currents, 38.9, 12.7, and 21.1% were BK-type channel currents in the neurons isolated from the young, adolescent, and adult rats, respectively. Furthermore, application of paxilline effectively prolonged the half-width of the action potential in pyramidal neurons in slices isolated from young and adult rats but not in neurons isolated from adolescent rats. We conclude that the availability of BK channel currents decreases in medial PFC pyramidal neurons of adolescent rats compared with those in the neurons of young and adult rats while their properties did not change across ages.

Postdecentralization plasticity of voltage-gated Na+ currents in rat glandular sympathetic neurons.

The kinetic properties of voltage-gated Na(+) currents in two groups of glandular postganglionic sympathetic neurons were assessed. The first group of neurons remained innervated by preganglionic axons until the day of current recordings, while the second--decentralized 4 weeks prior to recordings. An increase of maximum current amplitude and density was noted in decentralized neurons. Na(+) currents activated and time-dependently inactivated more slowly in decentralized than in control neurons. Furthermore, after decentralization the currents steady-state inactivated at less hyperpolarized potentials as well as reactivated faster from inactivation. We conclude that the Na(+) currents in decentralized postganglionic glandular sympathetic neurons undergo up-regulation.

β-Adrenergic receptor agonist increases voltage-gated Na(+) currents in medial prefrontal cortex pyramidal neurons.

The prefrontal cortex does not function properly in neuropsychiatric diseases and during chronic stress. The aim of this study was to test the effects of isoproterenol, a β-adrenergic receptor agonist, on the voltage-dependent fast-inactivating Na(+) currents in medial prefrontal cortex (mPFC) pyramidal neurons obtained from young rats. The recordings were performed in the cell-attached configuration. Isoproterenol (2μM) did not change the peak Na(+) current amplitude but shifted the IV curve of the Na(+) currents toward hyperpolarization. Pretreatment of the cells with the β-adrenergic antagonists propranolol and metoprolol abolished the effect of isoproterenol on the Na(+) currents, suggesting the involvement of β1-adrenergic receptors. The effect of β-adrenergic receptor stimulation on the sodium currents was dependent on kinase A and kinase C; the effect was diminished in the presence of the kinase A antagonist H-89 and the kinase C antagonist chelerythrine and abolished when the antagonists were coapplied. Moreover, isoproterenol depolarized the membrane potential recorded using the perforated-patch method, and this depolarization was abolished by cesium ions. Thus, in mPFC pyramidal neurons, stimulation of β-adrenergic receptors up-regulates the fast-inactivating voltage-gated Na(+) currents evoked by suprathreshold depolarizations.

Effects of ATP and GTP on voltage-gated K+ currents in glandular and muscular sympathetic neurons.

This study assesses the effects of ATP and GTP on the kinetic properties of voltage-gated K+ currents in anatomically identified postganglionic sympathetic neurons innervating the submandibular gland and the masseter muscle in rats. Three types of K+ currents were isolated: the I(Af) steady-state inactivating at more hyperpolarized potentials, I(As) steady-state inactivating at less hyperpolarized potentials than I(Af) and the I(K) current independent of membrane potential. The kinetic properties of these currents were tested in neurons with ATP (4 mM) and GTP (0.5 mM) or without ATP and GTP in the intracellular solution. In glandular and muscular neurons in the absence of ATP and GTP in the intracellular solution, the current density of I(Af) was significantly larger (142 pA/pF and 166 pA/pF, respectively) comparing to cells with ATP and GTP (96 pA/pF and 100 pA/pF, respectively). The I(As) was larger only in glandular neurons (52 pA/pF vs. 37 pA/pF).Conversely, I(K) current density was smaller in glandular and muscular neurons without ATP and GTP (17 pA/pF and 31 pA/pF, respectively) comparing to cells with ATP and GTP (57 pA/pF and 58 pA/pF, respectively). In glandular (15.5 nA/ms vs. 6.9 nA/ms) and muscular (10.9 nA/ms vs. 7.5 nA/ms) neurons, the I(Af) activated faster in the absence of ATP and GTP. Half inactivation voltage of I(Af) in glandular (-110.0 mV vs. -119.7 mV) and muscular (-108.4 vs. -117.3 mV) neurons was shifted towards depolarization in the absence of ATP and GTP. We suggest that the kinetic properties of K+ currents in glandular and muscular sympathetic neurons change markedly in the absence of ATP and GTP in the cytoplasm. Effectiveness of steady-state inactivated currents (I(Af) and I(AS)) increased, while effectiveness of steady-state noninactivated currents decreased in the absence of ATP and GTP. The effects were more pronounced in glandular than in muscular neurons.

Age‐dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na+ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20‐day old), late adolescent (60‐day old), and adult (6‐ to 7‐month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX‐resistant, low‐threshold, noninactivating, and voltage‐dependent Na+ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats.

Postdecentralization plasticity of voltage‐gated K+ currents in glandular sympathetic neurons in rats

This paper presents the kinetic and pharmacological properties of voltage‐gated K+ currents in anatomically identified glandular postganglionic sympathetic neurons isolated from the superior cervical ganglia in rats. The neurons were labelled by injecting the fluorescent tracer Fast Blue into the submandibular gland. The first group of neurons remained intact, i.e. innervated by the preganglionic axons until the day of current recordings (control neurons). The second group of neurons was denervated by severing the superior cervical trunk 4–6 weeks prior to current recordings (decentralized neurons). In every control and decentralized neuron three categories of voltage‐dependent K+ currents were found. (i) The IAf K+ current, steady state, inactivated at hyperpolarized membrane potentials. This current was fast activated and fast time‐dependently inactivated, insensitive to TEA and partially depressed by 4‐AP. (ii) The IAs K+ current, which was steady‐state inactivated at less hyperpolarized membrane potentials than IAf. The current activation and time‐dependent inactivation kinetics were slower than those of IAf. IAs was blocked by TEA and partially inhibited by 4‐AP. (iii) The IK K+ current did not undergo steady‐state inactivation. In decentralized compared to control neurons the maximum IAf K+ current density (at +50 mV) increased from 116.9 ± 8.2 to 189.0 ± 11.5 pA/pF, the 10–90% current rise time decreased from 2.3 to 0.7 ms and the recovery from inactivation was faster. Similarly, in decentralized compared to control neurons the maximum IAs K+ current density (at +50 mV) increased from 49.9 ± 3.5 to 74.3 ± 5.0 pA/pF, the 10–90% current rise time shortened from 29 to 16 ms and the recovery from inactivation of the current was also faster. The maximum density (at +50 mV) of IK in decentralized compared to control neurons decreased from 76.6 ± 3.9 to 60.7 ± 6.3 pA/pF. We suggest that the upregulation of voltage‐gated time‐dependently‐inactivated K+ currents and their faster recovery from inactivation serve to restrain the activity of glandular sympathetic neurons after decentralization.

KA-11, a novel pyrrolidine-2,5-dione derived broad-spectrum anticonvulsant: its antiepileptogenic, antinociceptive properties and in vitro characterization

Recently, compound KA-11 was identified as a promising candidate for a new broad-spectrum anticonvulsant. This compound revealed wide protective activity across the most important animal models of seizures such as the maximal electroshock test (MES), the subcutaneous pentylenetetrazole test ( scPTZ), and the six-hertz test (6 Hz, 32 mA). Importantly, KA-11 was devoid of acute neurological activity, which was assessed by applying the chimney test (TD50 value higher than 1500 mg/kg). The preliminary in vivo results confirmed favorable anticonvulsant and safety properties of KA-11. With the aim of further biological characterization of KA-11, in the current studies we evaluated its antiepileptogenic activity in the kindling model of epilepsy induced by repeated injection of PTZ in mice. Furthermore, we assessed the antinociceptive activity of KA-11 in several animal pain models. As a result, KA-11 (at all doses applied: 25, 50, and 100 mg/kg) significantly delayed the progression of kindling induced by repeated injection of PTZ in mice. Additionally, KA-11 revealed potent antinociceptive activity in the formalin-induced tonic pain and, importantly, in the oxaliplatin-induced neuropathic pain model in mice. Moreover, KA-11 did not induce motor deficits in the rotarod test. Patch-clamp experiments revealed that one of the mechanisms of action of KA-11 is inhibition of voltage-gated sodium currents. Compound KA-11 appeared to be safe in relation to hepatotoxic properties as no phospholipidosis induction was determined in HepG2 cells at 50 μM, and a small, statistically significant decrease of cell viability was observed only at the highest used dose of 100 μM. Moreover, KA-11 did not affect the function of CYP2D6. The aforementioned hybrid substance proved to penetrate the biological membranes in the in vitro permeability assays.