- Email: [email protected]
β-Adrenergic receptor agonist increases voltage-gated Na+ currents in medial prefrontal cortex pyramidal neurons
Neuroscience Letters 595 (2015) 87–93
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
-Adrenergic receptor agonist increases voltage-gated Na+ currents in medial prefrontal cortex pyramidal neurons Bartlomiej Szulczyk a,b,∗ a b
Department of Drug Technology and Pharmaceutical Biotechnology, The Medical University of Warsaw, Poland Department of Physiology and Pathophysiology, CEPT, The Medical University of Warsaw, Poland
h i g h l i g h t s • Isoproterenol does not influence maximal Na+ currents in mPFC pyramidal neurons. • -Adrenergic receptor agonist increases suprathreshold Na+ currents. • The effect is dependent on kinase A and kinase C.
a r t i c l e
i n f o
Article history: Received 22 February 2015 Received in revised form 29 March 2015 Accepted 8 April 2015 Available online 9 April 2015 Keywords: Medial prefrontal cortex -Adrenergic receptors Na+ currents Cell-attached Isoproterenol Protein kinase
a b s t r a c t 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 -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 (2 M) 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 -adrenergic antagonists propranolol and metoprolol abolished the effect of isoproterenol on the Na+ currents, suggesting the involvement of 1-adrenergic receptors. The effect of -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 -adrenergic receptors up-regulates the fast-inactivating voltage-gated Na+ currents evoked by suprathreshold depolarizations. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The prefrontal cortex does not function properly during acute and chronic stress; this dysfunction is associated with excessive noradrenaline release. PFC function is also impaired in neuropsychiatric disorders, for example, ADHD, depression and schizophrenia [9,20]. -Adrenergic receptors are G-protein coupled receptors that influence cellular effectors via second messengers [22,18]. A adrenergic agonist has been reported to enhance the calcium currents in the amygdala and the Ih currents in the cerebellum
∗ Correspondence to: Laboratory of Physiology and Pathophysiology, CEPT, The Medical University of Warsaw, Banacha 1B, 02-097, Poland. Tel.: +48 22 116 6163. E-mail address: [email protected] http://dx.doi.org/10.1016/j.neulet.2015.04.015 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[11,22]. -Adrenergic receptors in the PFC also influence working memory [19]. There are many reports showing that -adrenergic receptor stimulation enhances excitatory synaptic transmission in the brain. Isoproterenol enhances the evoked excitatory postsynaptic currents in the medial PFC [12,10]. Moreover, in hippocampal neurons, -adrenergic receptors must be activated during certain patterns of synaptic stimulation to induce LTP [13]. Furthermore, in the amygdala and in the hippocampus, -adrenergic receptor stimulation itself is sufficient to induce LTP [11]. The influence of neurotransmitters on fast voltage-gated Na+ channels has already been assessed in the whole-cell configuration. Serotonin receptor activation decreased the maximal amplitude of Na+ currents in the neurons of the PFC [4], and dopamine receptor stimulation inhibited the peak Na+ currents in the hippocampus [3].
88
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
The aim of this study was to elucidate the function of -adrenergic receptors in regulating voltage-dependent and fastinactivating Na+ currents in mPFC pyramidal neurons. 2. Materials and methods 2.1. Preparation of slices The experimental procedures used in this study adhered to the institutional and international guidelines on the ethical use of animals (II local ethical committee decision 1/2009). The experiments were performed on the neurons of 3-week-old male rats. Brain slices were prepared as previously described [24,16]. After the induction of deep anesthesia using ethyl chloride, the brains were removed and placed in a cold (0–4 ◦ C), sucrose-based solution [16,24]. Coronal slices (300 m thick) were prepared from the cerebral prefrontal tissue. The slices were incubated for 40 min in warm (34 ◦ C), oxygenated standard ACSF [24,16]. During the recordings, the slices were perfused with the same ACSF containing cesium ions (3 mM, see results) and blockers of GABAergic and glutaminergic transmission: 50 M picrotoxin, 10 M DNQX (6,7-dinitroquinoxaline2,3-dione) and 50 M AP-5 (DL-2-amino-5-phosphonopentanoic acid). 2.2. Recordings in the cell-attached configuration The slices were maintained at room temperature. Images of the neurons were recorded using DIC optics. The recordings were obtained from layer V pyramidal neurons in the infralimbic and prelimbic mPFC at a depth of 600–800 m from the cortical surface. The recordings were obtained using Multiclamp 700A and Digidata 1332A software (pClamp 9.0, Molecular Devices, CA, USA). The voltage-dependent Na+ currents were recorded from macropatches after the formation of a G seal in the cell-attached configuration. The pipette solution contained the following compounds (in mM): NaCl (120), KCl (5), CaCl2 (2), MgCl2 (2), HEPES (10), 4-aminopyridine (4-AP; 5) and tetraethylammonium chloride (TEA-Cl; 30) at a pH of 7.4 and an osmolality of 300 mOsm/kg H2 O. The pipette open-tip resistance was 7–10 M. The cell-attached patch recordings were filtered at 10 kHz (eight-pole Bessel filter) and sampled at 20 kHz. The patches were ruptured after the sodium current recordings were finished; then, the membrane potential was measured in the current-clamp configuration. All of the voltage levels shown in Figs. 2 and 3 were calculated relative to this membrane potential value in each individual cell. Conductance–voltage relationships were calculated from peak current–voltage relationships using the predicted reversal potential extrapolated from the linear portion of the IV relationship. Conductance–voltage plots were normalized and fitted with a Boltzmann equation [17,2, pClamp software v. 10]. Steady-state inactivation curves were also normalized and fitted with a Boltzmann equation. 2.3. Recordings in the perforated-patch configuration The membrane potentials were recorded under current-clamp conditions in the gramicidin perforated-patch recording mode [1]. The pipette solution contained the following compounds (in mM): potassium gluconate (105), KCl (20), HEPES-Na+ (10), ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid (0.1) and gramicidin (10–20 g/ml) at a pH of 7.25 and an osmolality of 280 mOsm/kg H2 O. The progress during membrane perforation was monitored by observing the slow gradual decrease in the access resistance. After the access resistance reached a steady level,
the membrane potential was recorded for at least 10 min for the control, and if the potential remained stable, the influence of isoproterenol was assessed. The access resistance was periodically evaluated, and the recording was discarded if the resistance rapidly decreased. The chemical compounds were delivered to the whole bath. All of the results presented in this paper are shown as the mean ± S.E.M. Paired and unpaired Students t-tests were used (GraphPad InStat software v3.06). 3. Results 3.1. The effect of isoproterenol on the membrane potential The treatment of pyramidal neurons with 2 M isoproterenol depolarized the membrane potential of mPFC pyramidal neurons, as measured using perforated-patch recordings (Fig. 1Aa, −64.9 ± 5.2 mV in the control vs. −62.6 ± 5.3 mV during isoproterenol treatment, n = 5). The average membrane potential change exerted by isoproterenol was +2.3 ± 0.2 mV (n = 5, paired t-test, p = 0.0004, Fig. 1Ac left bar). Isoproterenol may cause this depolarization by opening Ih channels or closing leak potassium channels [22,25]. These channels are blocked by cesium ions [6,5]. In the constant presence of cesium ions (3 mM) in the bath, 2 M isoproterenol exerted no effect on the membrane potential (−67.5 ± 1.4 mV in the control vs. −67.9 ± 1.5 mV in the presence of isoproterenol, average change −0.4 ± 0.4 mV, n = 8, paired t-test p = 0.46, Fig. 1Ab and Ac right bar). This finding suggests that isoproterenol depolarizes the membrane potential by opening Ih channels or closing K+ channels. To test whether Ih channels or leak potassium channels are involved in the effect of isoproterenol on the membrane potential, the recordings were obtained in the presence of the Ih channel blocker ZD 7228 (25 M) in the bath. When the Ih channels were blocked, isoproterenol exerted no effect on the membrane potential (−81.2 ± 3.4 mV in the control vs. −81.0 ± 3.5 mV during isoproterenol application, average change was 0.2 ± 0.4 mV, paired t-test, p = 0.67, n = 5). This finding suggests that isoproterenol depolarizes the membrane potential by opening Ih channels. In the cell-attached configuration, the membrane potential is not controlled. To ensure that the increase in the sodium current caused by isoproterenol was not due to the depolarization of the membrane potential, all recordings of sodium channels were obtained with Cs ions (3 mM) in the bath. 3.2. Isolation of fast-inactivating voltage-gated Na+ currents recorded in the cell-attached configuration Sodium currents were isolated in the same manner as in our previous reports [24]. Briefly, voltage steps from –20 mV to –100 mV (applied to the pipette), which were preceded by a +25mV pre-pulse, induced large capacitance transients with inward Na+ currents (Fig. 1Ba and Ca). When the pre-pulse amplitude was –65 mV (applied to the pipette), the same rectangular voltage steps induced only capacitance transients because the Na+ current was in an inactive steady state (Fig. 1Bb and Cb). Subtraction of the traces presented in Fig. 1Cb from the traces shown in Fig. 1Ca revealed pure fast-inactivating Na+ currents, as indicated in Fig. 1C(a–b). In Fig. 1C(a–b), only five sweeps are shown for clarity. Steady-state inactivation was assessed using a series of 600 ms pre-pulses (from +5 mV to −55 mV, applied to the pipette) using a test pulse of −65 mV lasting 20 ms. This method of Na+ current isolation was applied under control conditions and after 3 min of isoproterenol application. The IV or steady-state-inactivation relationships were recorded from eight
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
89
Fig. 1. Effect of isoproterenol on the membrane potential in mPFC pyramidal neurons. Isoproterenol depolarizes the membrane potential in mPFC pyramidal neurons. Aa – recording of the membrane potential, Ac – left bar shows the averaged effect (+2.3 ± 0.2 mV, n = 5, paired t-test, p = 0.0004). With Cs+ ions (3 mM) in the bath, isoproterenol does not influence the membrane potential. Ab – recording of the membrane potential, Ac – right bar shows the averaged effect (−0.4 ± 0.4 mV, n = 8, paired t-test p = 0,46). B and C Na+ current recording method. Explained in the results section. Da. The sodium current is not changed when 400 M cadmium ions are added to the pipette solution. Db. The sodium current is abolished by the presence of 1 M TTX in the pipette solution. Calibration applies to C and D.
to twelve times and the average recording was calculated. At the end of the recordings of the patch Na+ current, the patch membrane was ruptured, and the true membrane potential was measured in the current-clamp configuration (−66.1 ± 0.7 mV, n = 46). All of the applied voltage steps plotted on the horizontal axes in Figs. 2 and 3 are expressed relative to the potential measured after the cell was ruptured. Voltage-gated K+ currents were blocked by the inclusion of TEACl and 4-AP in the pipette solution. Calcium channels were not recorded in this study because when 1 M tetrodotoxin citrate was added to the pipette solution, no current was recorded (n = 12, Fig. 1Db). Moreover, with a calcium channel blocker (cadmium ions, 400 M) in the pipette, the amplitude of the Na+ currents was the same (26.9 ± 1.9 pA, n = 17) as without cadmium ions in the pipette (28.4 ± 1.7 pA, n = 59, unpaired t-test, p = 0.63, Fig. 1Da). The maximal amplitude of the sodium currents varied from patch to patch because the number of channels that was sampled
varied in the different recordings. The effect of the -adrenergic agonist was tested when the maximal amplitude of the sodium currents was above 15 pA. 3.3. Effect of ˇ-adrenergic receptor stimulation on voltage-gated Na+ currents The maximum Na+ currents recorded under the control conditions (38.6 ± 7.8 pA) and at the end of the 3 min of isoproterenol (2 M) treatment (36.7 ± 7.3 pA) were not significantly different (paired t-test, p > 0.24, n = 11, Fig. 2Aab). The IV relationships of the Na+ currents were also compared in the control conditions and after the isoproterenol treatment (Fig. 2Ba). After -adrenergic receptor stimulation, the suprathreshold voltage steps (dotted lines in Fig. 2Ba) induced larger Na+ currents than those of the control recordings. In other words, the Na+ current activation curves were shifted toward hyperpolarization in the presence of isoproterenol. The original
90
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
Fig. 2. Effect of isoproterenol on voltage-gated Na+ currents. Aa. Peak Na+ currents in the control condition and after isoproterenol application. Ab. The average maximum amplitude of the Na+ currents in the control and in the presence of isoproterenol is not significantly different. Ba. The effect of isoproterenol on the Na+ channel IV curve. Vertical dotted lines show that the suprathreshold Na+ currents are larger in the presence of isoproterenol. Bb. Examples of the suprathreshold Na+ currents in the control and after isoproterenol application. An example neuron is shown. Ca. Normalized sodium current conductance-voltage plots are shifted toward hyperpolarization in the presence of isoproterenol compared with the control, the same neuron as in Ba. The average shift was 5.2 ± 0.85 mV, paired t-test, p = 0.0001. An example neuron is shown. Cb. Normalized sodium current steady-state inactivation curves are not significantly influenced by isoproterenol. An example neuron is shown.
recordings of the Na+ currents evoked by –10 mV and –20 mV depolarization steps in the control and in the presence of isoproterenol are shown in Fig. 2Bb. To quantify the hyperpolarizing shift of the sodium channel IV curve after -adrenergic receptor stimulation, the IV relationships were converted to conductance plots, normalized and fitted to a Boltzmann curve (Fig. 2Ca). The V0.5 of the Na+ current activation was more negative during the isoproterenol treatment (−15.9 ± 2.1 mV) than in the recordings obtained under control conditions (−10.7 ± 1.9 mV, n = 11, average activation curve shift of 5.18 ± 0.85 mV, paired t-test, p = 0.0001, Fig. 2Ca). The activation slope factor (7.6 ± 0.5 in the control) did not change after the addition of the drug (7.5 ± 0.6, n = 11, paired t-test, p = 0.77, Fig. 2Ca). The higher dose of isoproterenol (100 M) did not influence maximal amplitude of the Na+ currents (data not shown) but shifted the Na+ channel activation curve toward hyperpolarization (average shift was 5.02 ± 1.4 mV, n = 5, paired t-test, p = 0,02). The magnitude of this shift was the same as in case of 2 M isoproterenol (5.18 ± 0.85, n = 11, see above, unpaired t-test, p = 0.92), which proves that 2 M isoproterenol exerted the maximal effect on Na+ channels. Thus, this dose was used throughout the study, except for the experiments described in this paragraph.
The sodium channel inactivation curve was slightly (but not significantly) influenced by isoproterenol. The V0,5 of inactivation was −43.3 ± 2.2 mV in the control recordings and −45.2 ± 1.5 mV after -adrenergic receptor stimulation (n = 7, paired t-test, p = 0.08, Fig. 2Cb).
3.4. Isoproterenol modulates the Na+ current activation curve via the protein kinase A and C transduction pathway With the constant presence of the -adrenergic antagonist in the bath (propranolol, 30–60 M), the V0.5 of activation was not changed during -adrenergic receptor activation; the V0.5 values were −11.3 ± 2.2 mV in the control and –11.5 ± 2.5 mV after the isoproterenol application (n = 6, paired t-test, p = 0.82, Fig. 3Aa). Because propranolol is a nonselective -adrenergic antagonist, similar experiments were performed in the presence of the selective 1-adrenergic antagonist metoprolol (60 M). With metoprolol in the bath, isoproterenol also did not shift the sodium channel activation curve. The V0,5 of activation was −9.5 ± 2.9 mV in the control and −10.5 ± 2.3 mV after the -adrenergic receptor stimulation (n = 4, Wilcoxon matched pairs test, p = 0.62, Fig. 3Ab). This finding indicates that the effect of the -adrenergic agonist depends on the type of 1 adrenergic receptor.
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
91
Fig. 3. Pharmacology of the effect of isoproterenol on voltage-gated Na+ currents. Aa. The normalized Na+ channel activation curve is not influenced by isoproterenol in the presence of propranolol. An example neuron is shown. Ab. The normalized Na+ channel activation curve is not influenced by isoproterenol in the presence of metoprolol. An example neuron is shown. Ba. The influence of isoproterenol on the normalized Na+ channel activation curve in the presence of H-89. An example neuron is shown. Bb. The average change in the V0,5 of activation when isoproterenol was coapplied with H-89 (left bar, 2.1 ± 0.6 n = 9) was significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.014). Ca. The influence of isoproterenol on the normalized Na+ channel activation curve in the presence of chelerythrine. An example neuron is shown. Cb. The average V0,5 of the activation shift when isoproterenol was coapplied with chelerythrine (left bar, 2.5 ± 0.6 mV, n = 9) was significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.027). D. With H-89 and chelerythrine in the bath, isoproterenol exerted no effect on Na+ currents. a – normalized activation curves. b – The average V0,5 of activation change when isoproterenol with H-89 and chelerythrine were coapplied was insignificant (left bar, 0.7 ± 0.6 mV, n = 7) and significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test p = 0.0017). An example neuron is shown.
92
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
To determine whether the effect of isoproterenol on Na+ currents was dependent on protein kinase A activation, the slices were preincubated with a protein kinase A antagonist, H-89 (15 M). With the constant presence of H-89 in the bath, the halfactivation of the Na+ current was –12.0 ± 1.1 mV in the control and −14.7 ± 0.9 mV after the -adrenergic agonist application (n = 9, Fig. 3Ba). This result means that in the presence of H-89, isoproterenol shifted the Na+ channel activation curve by 2.1 ± 0.6 mV. This shift was significantly smaller than when the effect of isoproterenol was tested without H-89 (the average activation curve shift without H-89 was 5.2 ± 0.8 mV, n = 11, data were taken from Fig. 2Ca, unpaired t-test, p = 0.014, Fig. 3Bb). Therefore, the PKA inhibitor diminished the effect of the -adrenergic receptor activation on the voltage-dependent Na+ currents in the mPFC pyramidal neurons. Additionally, the slices were preincubated with the kinase C antagonist chelerythrine (15 M). With chelerythrine in the bath, the -adrenergic receptor agonist also shifted the Na+ current activation curve toward hyperpolarization by 2.5 ± 0.6 mV (n = 9, Fig. 3Ca, V0,5 was −10.6 ± 2.1 mV in the control and −13.4 ± 1.7 mV after isoproterenol application). This effect was significantly weaker than when isoproterenol was applied without chelerythrine, as shown in Fig. 2Ca (2.5 ± 0.6 mV vs. 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.027, Fig. 3Ca and Cb). This finding shows that isoproterenol up-regulates sodium currents not only via kinase A but also via the kinase C transduction pathway. Furthermore, the effect of isoproterenol on the sodium current IV curve was tested in the presence of a kinase A antagonist (H-89, 15 M) and a kinase C antagonist (chelerythrine, 15 M), which were applied together to the bath. During the blockage of kinase A and C, the -adrenergic agonist did not influence the Na+ channel IV curve (the V0,5 was –9.9 ± 1.6 mV in the control and –10.6 ± 1.9 mV after the -adrenergic agonist application, n = 7, paired t-test, p = 0.3, Fig. 3Da and Db).
4. Discussion The recordings were performed from several sodium channels located in the patch membrane (macropatch). All sodium channels recordings were obtained in the presence of Cs+ ions to abolish the depolarization induced by isoproterenol (see results). In entorhinal cortex neurons and in PFC pyramidal neurons (cell-attached recordings), dopamine receptor activation shifted the Na+ current activation curve toward hyperpolarization, exactly as shown in this study [24,21]. Moreover, in ventricular myocyte macropatches, isoproterenol also up-regulated suprathreshold voltage-gated sodium channels, similar to this study [18]. Taken together, these results suggest that neurotransmitters may influence not only the peak amplitude of ionic channels but also the amplitudes in the range between the threshold and maximum. In this study, the effect of the -receptor agonist was diminished in the presence of kinase A and kinase C antagonists when they were applied separately and abolished when the inhibitors were coapplied, suggesting that only these two pathways are involved in the effect of isoproterenol on Na+ currents. It has been repeatedly reported that isoproterenol exerts its effects via kinase A stimulation [15,12,11]. For example, isoproterenol enhances the excitatory synaptic transmission in the visual cortex and PFC [15,12] and the calcium current in the amygdala [11], and these isoproterenol effects require kinase A activation. It has been shown that epinephrine acting on -adrenergic receptors potentiated the voltage-gated Na+ currents in DRG neurons, and this effect was mediated not only by kinase A but also by kinase C [14]. Interestingly, epinephrine enhanced the
maximal sodium currents and the sodium currents evoked by smaller depolarization steps, the same result as in this study [14]. Moreover, it was found that the activation curve of persistent Na+ channels was shifted toward hyperpolarization by dopamine, and this effect was mediated by protein kinase C [8]. Kinase C activation also shifted the activation curve of persistent Na+ channels toward more negative potentials in neocortical slices [2]. It has been reported that EPSPs are amplified by voltage-gated sodium channels in the PFC, in other cortical neurons and in the hippocampus [23,7,17,21]. It has been shown that the persistent Na+ current is responsible for EPSP amplification, but the fast Na+ current may also play a role in this phenomenon [7,17]. The question arises of how the up-regulation of sodium currents by isoproterenol observed in this study may contribute to the -adrenergic receptor-stimulated enhancement of the excitatory postsynaptic currents reported in the medial PFC [12,10]. The mechanism of this enhancement was increased glutamate release from presynaptic terminals [10,12] and increased NMDA receptor phosphorylation induced by a -adrenergic agonist [12]. One may suspect that the synaptic responses [10,12] could be further strengthened by increased EPSP amplification in the presence of isoproterenol. Because isoproterenol enhances the Na+ currents induced by small depolarizations (this study), postsynaptic responses [10,12] could activate a larger Na+ current when -adrenergic receptors are stimulated, thus potentially strengthening these postsynaptic responses. One may also suspect that the depolarization caused by isoproterenol (which is abolished in this study by cesium ions) may further strengthen EPSP amplification because the EPSP amplification caused by Na+ channels increases with membrane depolarization [7,23]. In conclusion, this study shows that isoproterenol up-regulates suprathreshold Na+ currents. One may suspect that this process modulates the cognitive functions of the PFC. Further behavioral studies are needed to determine the direction of this modulation. Certainly, the results presented in this study show that -adrenergic receptors modulate PFC function via voltage-gated sodium channels.
Acknowledgements I would like to thank Marta Kuzniarska and Izabela Zaborowska for their technical assistance. This study was sponsored by grants Nos. NN401584638 and NN301572940.
References [1] N. Akaike, N. Harata, Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms, Jpn. J. Physiol. 44 (1994) 433–473. [2] N. Astman, M.J. Gutnick, I.A. Fleidervish, Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices, J. Neurophysiol. 80 (1998) 1547–1551. [3] A.R. Cantrell, T. Scheuer, W.A. Catterall, Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons, J. Neurosci. 19 (1999) 5301–5310. [4] D.B. Carr, D.C. Cooper, S.L. Ulrich, N. Spruston, D.J. Surmeier, Serotonin receptor activation inhibits sodium current and dendritic excitability in prefrontal cortex via a protein kinase C-dependent mechanism, J. Neurosci. 22 (2002) 6846–6855. [5] Y. Dong, D. Cooper, F. Nasif, X.T. Hu, F.J.J. White, Dopamine modulates inwardly rectifying potassium currents in medial prefrontal cortex pyramidal neurons, J. Neurosci. 24 (2004) 3077–3085. [6] C.J. Giesbrecht, J.P. Mackay, H.B. Silveira, J.H. Urban, W.F. Colmers, Countervailing modulation of Ih by neuropeptide Y and corticotrophin-releasing factor in basolateral amygdala as a possible mechanism for their effects on stress-related behaviors, J. Neurosci. 30 (2010) 16970–16982. [7] G. Gonzalez-Burgos, G. Barrionuevo, Voltage-gated sodium channels shape subthreshold EPSPs in layer 5 pyramidal neurons from rat prefrontal cortex, J. Neurophysiol. 86 (2001) 1671–1684.
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93 [8] N.A. Gorelova, C.R. Yang, Dopamine D1/D5 receptor activation modulates a persistent sodium current in rat prefrontal cortical neurons in vitro, J. Neurophysiol. 84 (2000) 75–87. [9] A.B. Hains, A.F. Arnsten, Molecular mechanisms of stress-induced prefrontal cortical impairment: implications for mental illness, Learn. Memory 15 (2008) 551–564. [10] C.C. Huang, K.S. Hsu, Presynaptic mechanism underlying cAMP-induced synaptic potentiation in medial prefrontal cortex pyramidal neurons, Mol. Pharmacol. 69 (2006) 846–856. [11] C.C. Huang, S.J. Wang, P.W. Gean, Selective enhancement of P-type calcium currents by isoproterenol in the rat amygdala, J. Neurosci. 18 (1998) 2276–2282. [12] X.H. Ji, X.H. Cao, C.L. Zhang, Z.J. Feng, X.H. Zhang, L. Ma, B.M. Li, Pre- and postsynaptic beta-adrenergic activation enhances excitatory synaptic transmission in layer V/VI pyramidal neurons of the medial prefrontal cortex of rats, Cereb. Cortex. 18 (2008) 1506–1520. [13] H. Katsuki, Y. Izumi, C.F. Zorumski, Noradrenergic regulation of synaptic plasticity in the hippocampal CA1 region, J. Neurophysiol. 77 (1997) 3013–3020. [14] S.G. Khasar, G. McCarter, J.D. Levine, Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors, J. Neurophysiol. 81 (1999) 1104–1112. [15] M. Kobayashi, Differential regulation of synaptic transmission by adrenergic agonists via protein kinase A and protein kinase C in layer V pyramidal neurons of rat cerebral cortex, Neuroscience 146 (2007) 1772–1784. ˙ [16] A. Ksia˛ zek, W. Ładno, B. Szulczyk, K. Grzelka, P. Szulczyk, Properties of BK-type Ca+ (+)-dependent K+ channel currents in medial prefrontal cortex pyramidal neurons in rats of different ages, Front. Cell Neurosci. 7 (2013) 185.
93
[17] J.C. Magee, D. Johnston, Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons, J. Physiol. 487 (1995) 67–90. [18] K. Ono, H.A. Fozzard, D.A. Hanck, Mechanism of cAMP dependent modulation of cardiac sodium channel current kinetics, Circ. Res. 72 (1993) 807–815. [19] B.P. Ramos, L. Colgan, E. Nou, S. Ovadia, S.R. Wilson, A.F. Arnsten, The beta-1 adrenergic antagonist betaxolol improves working memory performance in rats and monkeys, Biol. Psychiatry 58 (2005) 894–900. [20] B.P. Ramos, A.F. Arnsten, Adrenergic pharmacology and cognition: focus on the prefrontal cortex, Pharmacol. Ther. 113 (2007) 523–536. [21] J.A. Rosenkranz, D. Johnston, State-dependent modulation of amygdala inputs by dopamine-induced enhancement of sodium currents in layer V enthorinal cortex, J. Neurosci. 27 (2007) 7054–7069. [22] F. Saitow, S. Konishi, Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells, J. Neurophysiol. 84 (2000) 2026–2034. [23] G. Stuart, B. Sakmann, Amplification of EPSPs by axosomatic sodium channels in neocortical pyramidal neurons, Neuron 15 (1995) 1065–1076. ˙ [24] B. Szulczyk, A. Ksia˛ zek, W. Ładno, P. Szulczyk, Effect of dopamine receptor stimulation on voltage-dependent fast-inactivating Na+ currents in medial prefrontal cortex (mPFC) pyramidal neurons in adult rats, Acta Neurobiol. Exp. (Wars.) 72 (2012) 351–364. [25] G. Witkowski, B. Szulczyk, R. Rola, P. Szulczyk, D(1) dopaminergic control of G protein-dependent inward rectifier K+ (GIRK)-like channel current in pyramidal neurons of the medial prefrontal cortex, Neuroscience 155 (2008) 53–63.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
-Adrenergic receptor agonist increases voltage-gated Na+ currents in medial prefrontal cortex pyramidal neurons Bartlomiej Szulczyk a,b,∗ a b
Department of Drug Technology and Pharmaceutical Biotechnology, The Medical University of Warsaw, Poland Department of Physiology and Pathophysiology, CEPT, The Medical University of Warsaw, Poland
h i g h l i g h t s • Isoproterenol does not influence maximal Na+ currents in mPFC pyramidal neurons. • -Adrenergic receptor agonist increases suprathreshold Na+ currents. • The effect is dependent on kinase A and kinase C.
a r t i c l e
i n f o
Article history: Received 22 February 2015 Received in revised form 29 March 2015 Accepted 8 April 2015 Available online 9 April 2015 Keywords: Medial prefrontal cortex -Adrenergic receptors Na+ currents Cell-attached Isoproterenol Protein kinase
a b s t r a c t 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 -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 (2 M) 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 -adrenergic antagonists propranolol and metoprolol abolished the effect of isoproterenol on the Na+ currents, suggesting the involvement of 1-adrenergic receptors. The effect of -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 -adrenergic receptors up-regulates the fast-inactivating voltage-gated Na+ currents evoked by suprathreshold depolarizations. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The prefrontal cortex does not function properly during acute and chronic stress; this dysfunction is associated with excessive noradrenaline release. PFC function is also impaired in neuropsychiatric disorders, for example, ADHD, depression and schizophrenia [9,20]. -Adrenergic receptors are G-protein coupled receptors that influence cellular effectors via second messengers [22,18]. A adrenergic agonist has been reported to enhance the calcium currents in the amygdala and the Ih currents in the cerebellum
∗ Correspondence to: Laboratory of Physiology and Pathophysiology, CEPT, The Medical University of Warsaw, Banacha 1B, 02-097, Poland. Tel.: +48 22 116 6163. E-mail address: [email protected] http://dx.doi.org/10.1016/j.neulet.2015.04.015 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
[11,22]. -Adrenergic receptors in the PFC also influence working memory [19]. There are many reports showing that -adrenergic receptor stimulation enhances excitatory synaptic transmission in the brain. Isoproterenol enhances the evoked excitatory postsynaptic currents in the medial PFC [12,10]. Moreover, in hippocampal neurons, -adrenergic receptors must be activated during certain patterns of synaptic stimulation to induce LTP [13]. Furthermore, in the amygdala and in the hippocampus, -adrenergic receptor stimulation itself is sufficient to induce LTP [11]. The influence of neurotransmitters on fast voltage-gated Na+ channels has already been assessed in the whole-cell configuration. Serotonin receptor activation decreased the maximal amplitude of Na+ currents in the neurons of the PFC [4], and dopamine receptor stimulation inhibited the peak Na+ currents in the hippocampus [3].
88
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
The aim of this study was to elucidate the function of -adrenergic receptors in regulating voltage-dependent and fastinactivating Na+ currents in mPFC pyramidal neurons. 2. Materials and methods 2.1. Preparation of slices The experimental procedures used in this study adhered to the institutional and international guidelines on the ethical use of animals (II local ethical committee decision 1/2009). The experiments were performed on the neurons of 3-week-old male rats. Brain slices were prepared as previously described [24,16]. After the induction of deep anesthesia using ethyl chloride, the brains were removed and placed in a cold (0–4 ◦ C), sucrose-based solution [16,24]. Coronal slices (300 m thick) were prepared from the cerebral prefrontal tissue. The slices were incubated for 40 min in warm (34 ◦ C), oxygenated standard ACSF [24,16]. During the recordings, the slices were perfused with the same ACSF containing cesium ions (3 mM, see results) and blockers of GABAergic and glutaminergic transmission: 50 M picrotoxin, 10 M DNQX (6,7-dinitroquinoxaline2,3-dione) and 50 M AP-5 (DL-2-amino-5-phosphonopentanoic acid). 2.2. Recordings in the cell-attached configuration The slices were maintained at room temperature. Images of the neurons were recorded using DIC optics. The recordings were obtained from layer V pyramidal neurons in the infralimbic and prelimbic mPFC at a depth of 600–800 m from the cortical surface. The recordings were obtained using Multiclamp 700A and Digidata 1332A software (pClamp 9.0, Molecular Devices, CA, USA). The voltage-dependent Na+ currents were recorded from macropatches after the formation of a G seal in the cell-attached configuration. The pipette solution contained the following compounds (in mM): NaCl (120), KCl (5), CaCl2 (2), MgCl2 (2), HEPES (10), 4-aminopyridine (4-AP; 5) and tetraethylammonium chloride (TEA-Cl; 30) at a pH of 7.4 and an osmolality of 300 mOsm/kg H2 O. The pipette open-tip resistance was 7–10 M. The cell-attached patch recordings were filtered at 10 kHz (eight-pole Bessel filter) and sampled at 20 kHz. The patches were ruptured after the sodium current recordings were finished; then, the membrane potential was measured in the current-clamp configuration. All of the voltage levels shown in Figs. 2 and 3 were calculated relative to this membrane potential value in each individual cell. Conductance–voltage relationships were calculated from peak current–voltage relationships using the predicted reversal potential extrapolated from the linear portion of the IV relationship. Conductance–voltage plots were normalized and fitted with a Boltzmann equation [17,2, pClamp software v. 10]. Steady-state inactivation curves were also normalized and fitted with a Boltzmann equation. 2.3. Recordings in the perforated-patch configuration The membrane potentials were recorded under current-clamp conditions in the gramicidin perforated-patch recording mode [1]. The pipette solution contained the following compounds (in mM): potassium gluconate (105), KCl (20), HEPES-Na+ (10), ethylene glycol-bis-(2-aminoethylether)-N,N,N ,N -tetraacetic acid (0.1) and gramicidin (10–20 g/ml) at a pH of 7.25 and an osmolality of 280 mOsm/kg H2 O. The progress during membrane perforation was monitored by observing the slow gradual decrease in the access resistance. After the access resistance reached a steady level,
the membrane potential was recorded for at least 10 min for the control, and if the potential remained stable, the influence of isoproterenol was assessed. The access resistance was periodically evaluated, and the recording was discarded if the resistance rapidly decreased. The chemical compounds were delivered to the whole bath. All of the results presented in this paper are shown as the mean ± S.E.M. Paired and unpaired Students t-tests were used (GraphPad InStat software v3.06). 3. Results 3.1. The effect of isoproterenol on the membrane potential The treatment of pyramidal neurons with 2 M isoproterenol depolarized the membrane potential of mPFC pyramidal neurons, as measured using perforated-patch recordings (Fig. 1Aa, −64.9 ± 5.2 mV in the control vs. −62.6 ± 5.3 mV during isoproterenol treatment, n = 5). The average membrane potential change exerted by isoproterenol was +2.3 ± 0.2 mV (n = 5, paired t-test, p = 0.0004, Fig. 1Ac left bar). Isoproterenol may cause this depolarization by opening Ih channels or closing leak potassium channels [22,25]. These channels are blocked by cesium ions [6,5]. In the constant presence of cesium ions (3 mM) in the bath, 2 M isoproterenol exerted no effect on the membrane potential (−67.5 ± 1.4 mV in the control vs. −67.9 ± 1.5 mV in the presence of isoproterenol, average change −0.4 ± 0.4 mV, n = 8, paired t-test p = 0.46, Fig. 1Ab and Ac right bar). This finding suggests that isoproterenol depolarizes the membrane potential by opening Ih channels or closing K+ channels. To test whether Ih channels or leak potassium channels are involved in the effect of isoproterenol on the membrane potential, the recordings were obtained in the presence of the Ih channel blocker ZD 7228 (25 M) in the bath. When the Ih channels were blocked, isoproterenol exerted no effect on the membrane potential (−81.2 ± 3.4 mV in the control vs. −81.0 ± 3.5 mV during isoproterenol application, average change was 0.2 ± 0.4 mV, paired t-test, p = 0.67, n = 5). This finding suggests that isoproterenol depolarizes the membrane potential by opening Ih channels. In the cell-attached configuration, the membrane potential is not controlled. To ensure that the increase in the sodium current caused by isoproterenol was not due to the depolarization of the membrane potential, all recordings of sodium channels were obtained with Cs ions (3 mM) in the bath. 3.2. Isolation of fast-inactivating voltage-gated Na+ currents recorded in the cell-attached configuration Sodium currents were isolated in the same manner as in our previous reports [24]. Briefly, voltage steps from –20 mV to –100 mV (applied to the pipette), which were preceded by a +25mV pre-pulse, induced large capacitance transients with inward Na+ currents (Fig. 1Ba and Ca). When the pre-pulse amplitude was –65 mV (applied to the pipette), the same rectangular voltage steps induced only capacitance transients because the Na+ current was in an inactive steady state (Fig. 1Bb and Cb). Subtraction of the traces presented in Fig. 1Cb from the traces shown in Fig. 1Ca revealed pure fast-inactivating Na+ currents, as indicated in Fig. 1C(a–b). In Fig. 1C(a–b), only five sweeps are shown for clarity. Steady-state inactivation was assessed using a series of 600 ms pre-pulses (from +5 mV to −55 mV, applied to the pipette) using a test pulse of −65 mV lasting 20 ms. This method of Na+ current isolation was applied under control conditions and after 3 min of isoproterenol application. The IV or steady-state-inactivation relationships were recorded from eight
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
89
Fig. 1. Effect of isoproterenol on the membrane potential in mPFC pyramidal neurons. Isoproterenol depolarizes the membrane potential in mPFC pyramidal neurons. Aa – recording of the membrane potential, Ac – left bar shows the averaged effect (+2.3 ± 0.2 mV, n = 5, paired t-test, p = 0.0004). With Cs+ ions (3 mM) in the bath, isoproterenol does not influence the membrane potential. Ab – recording of the membrane potential, Ac – right bar shows the averaged effect (−0.4 ± 0.4 mV, n = 8, paired t-test p = 0,46). B and C Na+ current recording method. Explained in the results section. Da. The sodium current is not changed when 400 M cadmium ions are added to the pipette solution. Db. The sodium current is abolished by the presence of 1 M TTX in the pipette solution. Calibration applies to C and D.
to twelve times and the average recording was calculated. At the end of the recordings of the patch Na+ current, the patch membrane was ruptured, and the true membrane potential was measured in the current-clamp configuration (−66.1 ± 0.7 mV, n = 46). All of the applied voltage steps plotted on the horizontal axes in Figs. 2 and 3 are expressed relative to the potential measured after the cell was ruptured. Voltage-gated K+ currents were blocked by the inclusion of TEACl and 4-AP in the pipette solution. Calcium channels were not recorded in this study because when 1 M tetrodotoxin citrate was added to the pipette solution, no current was recorded (n = 12, Fig. 1Db). Moreover, with a calcium channel blocker (cadmium ions, 400 M) in the pipette, the amplitude of the Na+ currents was the same (26.9 ± 1.9 pA, n = 17) as without cadmium ions in the pipette (28.4 ± 1.7 pA, n = 59, unpaired t-test, p = 0.63, Fig. 1Da). The maximal amplitude of the sodium currents varied from patch to patch because the number of channels that was sampled
varied in the different recordings. The effect of the -adrenergic agonist was tested when the maximal amplitude of the sodium currents was above 15 pA. 3.3. Effect of ˇ-adrenergic receptor stimulation on voltage-gated Na+ currents The maximum Na+ currents recorded under the control conditions (38.6 ± 7.8 pA) and at the end of the 3 min of isoproterenol (2 M) treatment (36.7 ± 7.3 pA) were not significantly different (paired t-test, p > 0.24, n = 11, Fig. 2Aab). The IV relationships of the Na+ currents were also compared in the control conditions and after the isoproterenol treatment (Fig. 2Ba). After -adrenergic receptor stimulation, the suprathreshold voltage steps (dotted lines in Fig. 2Ba) induced larger Na+ currents than those of the control recordings. In other words, the Na+ current activation curves were shifted toward hyperpolarization in the presence of isoproterenol. The original
90
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
Fig. 2. Effect of isoproterenol on voltage-gated Na+ currents. Aa. Peak Na+ currents in the control condition and after isoproterenol application. Ab. The average maximum amplitude of the Na+ currents in the control and in the presence of isoproterenol is not significantly different. Ba. The effect of isoproterenol on the Na+ channel IV curve. Vertical dotted lines show that the suprathreshold Na+ currents are larger in the presence of isoproterenol. Bb. Examples of the suprathreshold Na+ currents in the control and after isoproterenol application. An example neuron is shown. Ca. Normalized sodium current conductance-voltage plots are shifted toward hyperpolarization in the presence of isoproterenol compared with the control, the same neuron as in Ba. The average shift was 5.2 ± 0.85 mV, paired t-test, p = 0.0001. An example neuron is shown. Cb. Normalized sodium current steady-state inactivation curves are not significantly influenced by isoproterenol. An example neuron is shown.
recordings of the Na+ currents evoked by –10 mV and –20 mV depolarization steps in the control and in the presence of isoproterenol are shown in Fig. 2Bb. To quantify the hyperpolarizing shift of the sodium channel IV curve after -adrenergic receptor stimulation, the IV relationships were converted to conductance plots, normalized and fitted to a Boltzmann curve (Fig. 2Ca). The V0.5 of the Na+ current activation was more negative during the isoproterenol treatment (−15.9 ± 2.1 mV) than in the recordings obtained under control conditions (−10.7 ± 1.9 mV, n = 11, average activation curve shift of 5.18 ± 0.85 mV, paired t-test, p = 0.0001, Fig. 2Ca). The activation slope factor (7.6 ± 0.5 in the control) did not change after the addition of the drug (7.5 ± 0.6, n = 11, paired t-test, p = 0.77, Fig. 2Ca). The higher dose of isoproterenol (100 M) did not influence maximal amplitude of the Na+ currents (data not shown) but shifted the Na+ channel activation curve toward hyperpolarization (average shift was 5.02 ± 1.4 mV, n = 5, paired t-test, p = 0,02). The magnitude of this shift was the same as in case of 2 M isoproterenol (5.18 ± 0.85, n = 11, see above, unpaired t-test, p = 0.92), which proves that 2 M isoproterenol exerted the maximal effect on Na+ channels. Thus, this dose was used throughout the study, except for the experiments described in this paragraph.
The sodium channel inactivation curve was slightly (but not significantly) influenced by isoproterenol. The V0,5 of inactivation was −43.3 ± 2.2 mV in the control recordings and −45.2 ± 1.5 mV after -adrenergic receptor stimulation (n = 7, paired t-test, p = 0.08, Fig. 2Cb).
3.4. Isoproterenol modulates the Na+ current activation curve via the protein kinase A and C transduction pathway With the constant presence of the -adrenergic antagonist in the bath (propranolol, 30–60 M), the V0.5 of activation was not changed during -adrenergic receptor activation; the V0.5 values were −11.3 ± 2.2 mV in the control and –11.5 ± 2.5 mV after the isoproterenol application (n = 6, paired t-test, p = 0.82, Fig. 3Aa). Because propranolol is a nonselective -adrenergic antagonist, similar experiments were performed in the presence of the selective 1-adrenergic antagonist metoprolol (60 M). With metoprolol in the bath, isoproterenol also did not shift the sodium channel activation curve. The V0,5 of activation was −9.5 ± 2.9 mV in the control and −10.5 ± 2.3 mV after the -adrenergic receptor stimulation (n = 4, Wilcoxon matched pairs test, p = 0.62, Fig. 3Ab). This finding indicates that the effect of the -adrenergic agonist depends on the type of 1 adrenergic receptor.
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
91
Fig. 3. Pharmacology of the effect of isoproterenol on voltage-gated Na+ currents. Aa. The normalized Na+ channel activation curve is not influenced by isoproterenol in the presence of propranolol. An example neuron is shown. Ab. The normalized Na+ channel activation curve is not influenced by isoproterenol in the presence of metoprolol. An example neuron is shown. Ba. The influence of isoproterenol on the normalized Na+ channel activation curve in the presence of H-89. An example neuron is shown. Bb. The average change in the V0,5 of activation when isoproterenol was coapplied with H-89 (left bar, 2.1 ± 0.6 n = 9) was significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.014). Ca. The influence of isoproterenol on the normalized Na+ channel activation curve in the presence of chelerythrine. An example neuron is shown. Cb. The average V0,5 of the activation shift when isoproterenol was coapplied with chelerythrine (left bar, 2.5 ± 0.6 mV, n = 9) was significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.027). D. With H-89 and chelerythrine in the bath, isoproterenol exerted no effect on Na+ currents. a – normalized activation curves. b – The average V0,5 of activation change when isoproterenol with H-89 and chelerythrine were coapplied was insignificant (left bar, 0.7 ± 0.6 mV, n = 7) and significantly smaller than when only isoproterenol was applied (right bar, 5.2 ± 0.8 mV, n = 11, unpaired t-test p = 0.0017). An example neuron is shown.
92
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93
To determine whether the effect of isoproterenol on Na+ currents was dependent on protein kinase A activation, the slices were preincubated with a protein kinase A antagonist, H-89 (15 M). With the constant presence of H-89 in the bath, the halfactivation of the Na+ current was –12.0 ± 1.1 mV in the control and −14.7 ± 0.9 mV after the -adrenergic agonist application (n = 9, Fig. 3Ba). This result means that in the presence of H-89, isoproterenol shifted the Na+ channel activation curve by 2.1 ± 0.6 mV. This shift was significantly smaller than when the effect of isoproterenol was tested without H-89 (the average activation curve shift without H-89 was 5.2 ± 0.8 mV, n = 11, data were taken from Fig. 2Ca, unpaired t-test, p = 0.014, Fig. 3Bb). Therefore, the PKA inhibitor diminished the effect of the -adrenergic receptor activation on the voltage-dependent Na+ currents in the mPFC pyramidal neurons. Additionally, the slices were preincubated with the kinase C antagonist chelerythrine (15 M). With chelerythrine in the bath, the -adrenergic receptor agonist also shifted the Na+ current activation curve toward hyperpolarization by 2.5 ± 0.6 mV (n = 9, Fig. 3Ca, V0,5 was −10.6 ± 2.1 mV in the control and −13.4 ± 1.7 mV after isoproterenol application). This effect was significantly weaker than when isoproterenol was applied without chelerythrine, as shown in Fig. 2Ca (2.5 ± 0.6 mV vs. 5.2 ± 0.8 mV, n = 11, unpaired t-test, p = 0.027, Fig. 3Ca and Cb). This finding shows that isoproterenol up-regulates sodium currents not only via kinase A but also via the kinase C transduction pathway. Furthermore, the effect of isoproterenol on the sodium current IV curve was tested in the presence of a kinase A antagonist (H-89, 15 M) and a kinase C antagonist (chelerythrine, 15 M), which were applied together to the bath. During the blockage of kinase A and C, the -adrenergic agonist did not influence the Na+ channel IV curve (the V0,5 was –9.9 ± 1.6 mV in the control and –10.6 ± 1.9 mV after the -adrenergic agonist application, n = 7, paired t-test, p = 0.3, Fig. 3Da and Db).
4. Discussion The recordings were performed from several sodium channels located in the patch membrane (macropatch). All sodium channels recordings were obtained in the presence of Cs+ ions to abolish the depolarization induced by isoproterenol (see results). In entorhinal cortex neurons and in PFC pyramidal neurons (cell-attached recordings), dopamine receptor activation shifted the Na+ current activation curve toward hyperpolarization, exactly as shown in this study [24,21]. Moreover, in ventricular myocyte macropatches, isoproterenol also up-regulated suprathreshold voltage-gated sodium channels, similar to this study [18]. Taken together, these results suggest that neurotransmitters may influence not only the peak amplitude of ionic channels but also the amplitudes in the range between the threshold and maximum. In this study, the effect of the -receptor agonist was diminished in the presence of kinase A and kinase C antagonists when they were applied separately and abolished when the inhibitors were coapplied, suggesting that only these two pathways are involved in the effect of isoproterenol on Na+ currents. It has been repeatedly reported that isoproterenol exerts its effects via kinase A stimulation [15,12,11]. For example, isoproterenol enhances the excitatory synaptic transmission in the visual cortex and PFC [15,12] and the calcium current in the amygdala [11], and these isoproterenol effects require kinase A activation. It has been shown that epinephrine acting on -adrenergic receptors potentiated the voltage-gated Na+ currents in DRG neurons, and this effect was mediated not only by kinase A but also by kinase C [14]. Interestingly, epinephrine enhanced the
maximal sodium currents and the sodium currents evoked by smaller depolarization steps, the same result as in this study [14]. Moreover, it was found that the activation curve of persistent Na+ channels was shifted toward hyperpolarization by dopamine, and this effect was mediated by protein kinase C [8]. Kinase C activation also shifted the activation curve of persistent Na+ channels toward more negative potentials in neocortical slices [2]. It has been reported that EPSPs are amplified by voltage-gated sodium channels in the PFC, in other cortical neurons and in the hippocampus [23,7,17,21]. It has been shown that the persistent Na+ current is responsible for EPSP amplification, but the fast Na+ current may also play a role in this phenomenon [7,17]. The question arises of how the up-regulation of sodium currents by isoproterenol observed in this study may contribute to the -adrenergic receptor-stimulated enhancement of the excitatory postsynaptic currents reported in the medial PFC [12,10]. The mechanism of this enhancement was increased glutamate release from presynaptic terminals [10,12] and increased NMDA receptor phosphorylation induced by a -adrenergic agonist [12]. One may suspect that the synaptic responses [10,12] could be further strengthened by increased EPSP amplification in the presence of isoproterenol. Because isoproterenol enhances the Na+ currents induced by small depolarizations (this study), postsynaptic responses [10,12] could activate a larger Na+ current when -adrenergic receptors are stimulated, thus potentially strengthening these postsynaptic responses. One may also suspect that the depolarization caused by isoproterenol (which is abolished in this study by cesium ions) may further strengthen EPSP amplification because the EPSP amplification caused by Na+ channels increases with membrane depolarization [7,23]. In conclusion, this study shows that isoproterenol up-regulates suprathreshold Na+ currents. One may suspect that this process modulates the cognitive functions of the PFC. Further behavioral studies are needed to determine the direction of this modulation. Certainly, the results presented in this study show that -adrenergic receptors modulate PFC function via voltage-gated sodium channels.
Acknowledgements I would like to thank Marta Kuzniarska and Izabela Zaborowska for their technical assistance. This study was sponsored by grants Nos. NN401584638 and NN301572940.
References [1] N. Akaike, N. Harata, Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms, Jpn. J. Physiol. 44 (1994) 433–473. [2] N. Astman, M.J. Gutnick, I.A. Fleidervish, Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices, J. Neurophysiol. 80 (1998) 1547–1551. [3] A.R. Cantrell, T. Scheuer, W.A. Catterall, Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons, J. Neurosci. 19 (1999) 5301–5310. [4] D.B. Carr, D.C. Cooper, S.L. Ulrich, N. Spruston, D.J. Surmeier, Serotonin receptor activation inhibits sodium current and dendritic excitability in prefrontal cortex via a protein kinase C-dependent mechanism, J. Neurosci. 22 (2002) 6846–6855. [5] Y. Dong, D. Cooper, F. Nasif, X.T. Hu, F.J.J. White, Dopamine modulates inwardly rectifying potassium currents in medial prefrontal cortex pyramidal neurons, J. Neurosci. 24 (2004) 3077–3085. [6] C.J. Giesbrecht, J.P. Mackay, H.B. Silveira, J.H. Urban, W.F. Colmers, Countervailing modulation of Ih by neuropeptide Y and corticotrophin-releasing factor in basolateral amygdala as a possible mechanism for their effects on stress-related behaviors, J. Neurosci. 30 (2010) 16970–16982. [7] G. Gonzalez-Burgos, G. Barrionuevo, Voltage-gated sodium channels shape subthreshold EPSPs in layer 5 pyramidal neurons from rat prefrontal cortex, J. Neurophysiol. 86 (2001) 1671–1684.
B. Szulczyk / Neuroscience Letters 595 (2015) 87–93 [8] N.A. Gorelova, C.R. Yang, Dopamine D1/D5 receptor activation modulates a persistent sodium current in rat prefrontal cortical neurons in vitro, J. Neurophysiol. 84 (2000) 75–87. [9] A.B. Hains, A.F. Arnsten, Molecular mechanisms of stress-induced prefrontal cortical impairment: implications for mental illness, Learn. Memory 15 (2008) 551–564. [10] C.C. Huang, K.S. Hsu, Presynaptic mechanism underlying cAMP-induced synaptic potentiation in medial prefrontal cortex pyramidal neurons, Mol. Pharmacol. 69 (2006) 846–856. [11] C.C. Huang, S.J. Wang, P.W. Gean, Selective enhancement of P-type calcium currents by isoproterenol in the rat amygdala, J. Neurosci. 18 (1998) 2276–2282. [12] X.H. Ji, X.H. Cao, C.L. Zhang, Z.J. Feng, X.H. Zhang, L. Ma, B.M. Li, Pre- and postsynaptic beta-adrenergic activation enhances excitatory synaptic transmission in layer V/VI pyramidal neurons of the medial prefrontal cortex of rats, Cereb. Cortex. 18 (2008) 1506–1520. [13] H. Katsuki, Y. Izumi, C.F. Zorumski, Noradrenergic regulation of synaptic plasticity in the hippocampal CA1 region, J. Neurophysiol. 77 (1997) 3013–3020. [14] S.G. Khasar, G. McCarter, J.D. Levine, Epinephrine produces a beta-adrenergic receptor-mediated mechanical hyperalgesia and in vitro sensitization of rat nociceptors, J. Neurophysiol. 81 (1999) 1104–1112. [15] M. Kobayashi, Differential regulation of synaptic transmission by adrenergic agonists via protein kinase A and protein kinase C in layer V pyramidal neurons of rat cerebral cortex, Neuroscience 146 (2007) 1772–1784. ˙ [16] A. Ksia˛ zek, W. Ładno, B. Szulczyk, K. Grzelka, P. Szulczyk, Properties of BK-type Ca+ (+)-dependent K+ channel currents in medial prefrontal cortex pyramidal neurons in rats of different ages, Front. Cell Neurosci. 7 (2013) 185.
93
[17] J.C. Magee, D. Johnston, Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons, J. Physiol. 487 (1995) 67–90. [18] K. Ono, H.A. Fozzard, D.A. Hanck, Mechanism of cAMP dependent modulation of cardiac sodium channel current kinetics, Circ. Res. 72 (1993) 807–815. [19] B.P. Ramos, L. Colgan, E. Nou, S. Ovadia, S.R. Wilson, A.F. Arnsten, The beta-1 adrenergic antagonist betaxolol improves working memory performance in rats and monkeys, Biol. Psychiatry 58 (2005) 894–900. [20] B.P. Ramos, A.F. Arnsten, Adrenergic pharmacology and cognition: focus on the prefrontal cortex, Pharmacol. Ther. 113 (2007) 523–536. [21] J.A. Rosenkranz, D. Johnston, State-dependent modulation of amygdala inputs by dopamine-induced enhancement of sodium currents in layer V enthorinal cortex, J. Neurosci. 27 (2007) 7054–7069. [22] F. Saitow, S. Konishi, Excitability increase induced by beta-adrenergic receptor-mediated activation of hyperpolarization-activated cation channels in rat cerebellar basket cells, J. Neurophysiol. 84 (2000) 2026–2034. [23] G. Stuart, B. Sakmann, Amplification of EPSPs by axosomatic sodium channels in neocortical pyramidal neurons, Neuron 15 (1995) 1065–1076. ˙ [24] B. Szulczyk, A. Ksia˛ zek, W. Ładno, P. Szulczyk, Effect of dopamine receptor stimulation on voltage-dependent fast-inactivating Na+ currents in medial prefrontal cortex (mPFC) pyramidal neurons in adult rats, Acta Neurobiol. Exp. (Wars.) 72 (2012) 351–364. [25] G. Witkowski, B. Szulczyk, R. Rola, P. Szulczyk, D(1) dopaminergic control of G protein-dependent inward rectifier K+ (GIRK)-like channel current in pyramidal neurons of the medial prefrontal cortex, Neuroscience 155 (2008) 53–63.