Neuroscience Letters 559 (2014) 30–33
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Persistent sodium current properties in hippocampal CA1 pyramidal neurons of young and adult rats Oleksii Lunko a , Dmytro Isaev a,b , Oleksandr Maximyuk a,b , Gleb Ivanchick a , Vadym Sydorenko a , Oleg Krishtal a,b , Elena Isaeva a,∗ a b
Department of Cellular Membranology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine State Key Laboratory for Molecular and Cellular Biology, Kiev 01024, Ukraine
h i g h l i g h t s • • • • •
Persistent sodium current is an important contributor to cellular and network excitability. We analyzed changes in INaP properties during maturation. CA1 pyramidal cells were isolated from hippocampus of P12-16 and P60-75 rats. INaP density is substantially increased in the adult group. Hyperpolarization shift of INaP activation with increasing postnatal age.
a r t i c l e
i n f o
Article history: Received 12 July 2013 Received in revised form 14 November 2013 Accepted 18 November 2013 Keywords: Persistent tetrodotoxin-sensitive sodium current Hippocampal CA1 pyramidal neurons Postnatal development
a b s t r a c t Persistent tetrodotoxin-sensitive sodium current (INaP ) plays an important role in cellular and neuronal network excitability in physiological conditions and under different pathological circumstances. However, developmental changes in INaP properties remain largely unclear. In the present study using whole cell patch clamp technique we evaluated INaP properties in CA1 hippocampal pyramidal neurons isolated from young (postnatal day (P) 12–16) and adult (P60-75) rats. We show that the INaP density is substantially larger in the adult group. Although INaP inactivation characteristics were found to be similar in both groups, voltage dependence of INaP activation is shifted to more negative membrane potentials (young: −48.6 ± 0.5 mV vs. adult: −52.4 ± 0.2 mV, p < 0.01). Our data indicates the increase of INaP contribution in the basal membrane sodium conductivity in the mature hippocampus. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Slowly inactivating “persistent” Na+ current (INaP ) mediated by sodium channels with activation near the resting membrane potential was found in various central and peripheral neurons including neocortex, entorhinal cortex, thalamus, cerebellum, hippocampus and dorsal root ganglia (see [1,2] for ref). INaP plays an important role in subthreshold oscillations of membrane potential and sustains recurrent firing in response to membrane depolarization [3–5]. Moreover, INaP can amplify synaptic potentials and contributes to intrinsic pacemaking activity of different neurons [6–8]. Several reports indicate that pathological conditions, such as chronic epilepsy and hypoxia, can be accompanied by
∗ Corresponding author at: Department of Cellular Membranology, Bogomoletz Institute of Physiology, Bogomoletz str. 4, Kiev 01024, Ukraine. Tel.: +38 044 256 2519; fax: +38 044 256 2000. E-mail address: [email protected] (E. Isaeva). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.035
enhancement of INaP conductance implying involvement of this current in pathological synchronization [9–11]. Despite the significant role of INaP in the neuronal function little is known about age dependent changes in its properties. During last three decades after discovering of INaP only few studies were devoted to investigation of developmental changes in the expression of this current. Alzheimer with colleagues [12] showed threefold increase in the INaP density during first three postnatal weeks in pyramidal neurons of sensorimotor cortex. Authors suggested that INaP plays an important role in the control of excitability during early postnatal development. This data is in agreement with our previous report where activation of INaP substantially facilitated seizure-like activity in immature hippocampus [13]. However, developmental changes in INaP properties through the late adolescent period remain unclear. In the present study, patch clamp recordings were made from hippocampal CA1 pyramidal cells isolated from rats at the second postnatal week (postnatal days (P) 12–16) and at P60-75. Here we demonstrate substantial changes in both current density and steady-state activation of INaP during maturation.
O. Lunko et al. / Neuroscience Letters 559 (2014) 30–33
2. Materials and methods All experimental procedures were performed in accordance with the guidelines set by the National Institutes of Health for the humane treatment of animals and approved by the Animal Care Committee of Bogomoletz Institute of Physiology. 2.1. Slices and acutely isolated neurons preparations The hippocampal slice preparation and neuron dissociation procedure was performed as described previously with some modifications [14]. Briefly, young (P12-16, N = 19) and adult (P60-75, N = 23) Wistar rats were anesthetized with sevoflurane, decapitated and brain was rapidly moved into ice-cold oxygenated (95% O2 , 5% CO2 ) dissecting solution contained (in mM): 130 NaCl, 5 KCl, 0.1 CaCl2 , 5 MgCl2 , 1 NaH2 PO4 , 1 Na2 HPO4 , 26 NaHCO3 , 10 glucose. Hippocampal slices (400–500 m) were cut using a vibratome (MA752, Campden Instrument, Loughborough, UK) and kept for at least an hour at room temperature (22–24 ◦ C) in incubation solution contained (in mM): 130 NaCl, 5 KCl, 2 CaCl2 , 2 MgCl2 , 1 NaH2 PO4 , 1 Na2 HPO4 , 26 NaHCO3 , 10 glucose. Slices were then transferred to sucrose-based solution contained (in mM): 290 sucrose, 3 KCl, 0.5 CaCl2 , 2 MgCl2 , 10 HEPES, 15 glucose, 2 mg/ml pronase E (32 ◦ C). After 15 min of enzymatic treatment slices were rinsed with artificial cerebrospinal fluid (ACSF) contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2 , 2 MgCl2 , 20 HEPES (pH adjusted to 7.4 by NaOH). Isolation of neurons from pyramidal CA1 region of hippocampus was performed using vibrodissociation technique [14].
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Obtained data were summarized and fitted with mono exponential function. 2.3. Data analysis Statistical data analysis was performed using Prism 5 (GraphPad, La Jolla, CA) and Origin 7.5 (OriginLab, Northampton, MA) software. The Shapiro–Wilk test was used to estimate normality of the data for each group. Statistical analysis of the development changes in INaP amplitude and density was performed using unpaired twosample Student’s t-test. Extra sum-of-squares F-test was used for voltage dependence of INaP conductance fit comparison. All data in the text were presented as the mean ± SE. 2.4. Chemicals TTX was obtained from Tocris (Ellisville, MO). All other chemicals were purchased from Sigma (St. Louis, MO). 3. Results Whole cell INaP were elicited in isolated hippocampal CA1 pyramidal cells of young (P12-16) and adult (60–75) rats using slow ramp depolarization from holding potential −80 mV to 0 mV (30 mV/s) [13]. Fig. 1A shows five superimposed responses evoked
2.2. Electrophysiological recordings Conventional patch clamp technique in the whole cell configuration was used for the recording of INaP in acutely isolated pyramidal neurons. Extracellular solution consisted (in mM): 130 NaCl, 2 CaCl2 , 1.3 MgCl2 , 20 TEACl, 10 HEPES and 0.4 CdCl2 (pH was adjusted to 7.35 with NaOH). Patch pipettes were filled with an intracellular solution contained (in mM): 120 CsF, 5 NaCl, 30 TEACl, 10 EGTA, 10 Tris-HCl (pH was adjusted to 7.2 with CsOH). Pipette resistances were ranged from 3 to 5 M. All recordings were made using model 2400 patch clamp amplifier (A-M Systems, Carlsborg, WA). Recordings were digitized at 2 kHz by analogue-to-digital converter (NI PCI-6221, National instruments, Austin, TX, USA) and filtered offline for further analysis. Liquid junction potential was determined and command voltages were corrected accordingly. All experiments were made at the room temperature. INaP was elicited by slow depolarizing ramp voltage protocol (from −80 mV to 0 mV, 30 mV/s). Current–voltage relationships of INaP were obtained by subtraction of five averaged recordings before and after treatment with 1 M tetrodotoxin (TTX). The capacitance of cells was estimated from the change in membrane charge, determined from the integrated capacity transients evoked by a 10 mV hyperpolarizing step from a holding potential (Vh ) of −80 mV. Voltage dependence of INaP conductance (G) was calculated from I/(V − Vrev ), where I is the amplitude of INaP during the test depolarization (V), and Vrev is the Na+ reversal potential calculated from the Nernst equation for the given extra- and intracellular Na+ concentrations (82 mV throughout). To evaluate the voltage-dependence of INaP steadystate inactivation the conditioning prepulses varied from −80 to −10 mV (10 s duration, 10 mV step) were preceded each standard voltage ramp stimulation to elicit INaP . All voltage-dependence relationships were normalized to the maximal conductance for each recording, then summarized for each group and fitted with a Boltzmann function. The time-dependence of inactivation was evaluated by applying conditioning prepulses to −10 mV at various duration (from 100 ms to 10 s) before a voltage ramp protocol eliciting INaP .
Fig. 1. Persistent tetrodotoxin-sensitive Na+ current in hippocampal CA1 pyramidal neurons. (A) Representative averaged currents of five superimposed traces evoked by ramp voltage depolarization from −80 mV to 0 mV before and after exposure to 1 M TTX. (B) Examples of INaP measured in hippocampal CA1 pyramidal neurons isolated from P12 (1) and P67 (2) rats (current traces were obtained by subtraction of TTX resistant current from total current).
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O. Lunko et al. / Neuroscience Letters 559 (2014) 30–33
−52.4 ± 0.2 mV (n = 26) in adult group, p < 0.01). Next we examined inactivation properties of INaP for both ages. Fig. 2B,C demonstrates that the voltage dependence of INaP inactivation (V1/2 : −43.5 ± 2.4 mV (n = 5) in young vs. −43.2 ± 2.7 mV (n = 6) in adult group) as well as INaP inactivation onset ( decay : 3.1 ± 0.3 s in young (n = 7) vs. 2.9 ± 0.4 s (n = 8) in adult group) is similar for both groups suggesting that the ageing does not affect inactivation properties of INaP . 4. Discussion
Fig. 2. Effect of age on the gating properties of persistent Na+ current. (A) Averaged steady-state activation curves (G/Gmax ) were fitted by Boltzmann equation for both young (white) and adult (grey) rats. The activation curve in the adult group was shifted by 3.8 ± 0.7 mV to more negative membrane potentials as compared to the young group. (B) Summarized data for voltage-dependent inactivation revealed no changes in the steady-state inactivation of INaP between young and adult rats. (C) Time dependence of INaP inactivation was found to be virtually the same for both ages. Stimulation protocols for (B) and (C) are shown in the corresponding insets.
by the ramp protocol in the absence and presence of 1 M TTX recorded from CA1 pyramidal cell of P12 rat. Subtraction of corresponding TTX data from each recording resulted in IV curves of INaP (Fig. 1B). INaP was activated at near −70 mV and reached its peak between −40 mV and −30 mV. The peak INaP amplitude was substantially higher in the adult group (adult: 65.5 ± 6.8 pA (n = 26); young: 35.4 ± 4.7 pA (n = 16), p < 0.01). The likely cause of such changes could be an increase in the INaP current density in adult animals. Indeed, the INaP density expressed as the ratio of the peak INaP amplitude to cell capacitance was significantly higher in the adult group (adult: 3.1 ± 0.2 pA/pF (n = 26) vs young: 1.8 ± 0.2 pA/pF (n = 16); p < 0.01). The cell capacitance was found to be virtually the same for both groups (young: 20.0 ± 1.1 pF; adult: 20.5 ± 1.4 pF, p = 0.8). Fig. 2A illustrates developmental changes in the steady-state activation of INaP . While the slope factor was unchanged (young: 5.6 ± 0.4 (n = 16) vs in adult: 5.5 ± 0.1 (n = 26), p = 0.7), the steadystate current activation curve in the adult group was shifted by 3.8 ± 0.7 mV to more negative membrane potentials as compared to the young group (−48.6 ± 0.5 mV in young (n = 16) vs.
The main findings of the present study are significant increase in the INaP density and hyperpolarization shift of activation of Na+ channel responsible for INaP in CA1 hippocampal neurons during maturation. Thus, contribution of persistent Na+ conductance in this region is larger in mature brain: INaP amplitude is increased and this current becomes manifested at lower depolarization voltages. It was shown that in hippocampal CA1 pyramidal cells most of Na+ channels responsible for INaP located at or near the somata [15]. To preserve natural cell maturation in the present study we used isolated neurons consisted somata with small dendritic arborizations. So we could not exclude a possibility that a fraction of dendritic Na+ channels contributing to INaP could have different course of maturation. The increase in INaP amplitude during postnatal development cannot be attributed to changes in the membrane capacitance or in inactivation characteristics since these parameters were similar for both age groups. However, the density of INaP was considerably increased in the adult group. This increase in the INaP conductance could be attributed to a number of mechanisms, including increase in the expression or changes in a subunit composition of Na+ channel, which could lead to changes in channel properties, the increase or decrease in the influence of endogenous factors which can also modulate INaP properties [13,16,17]. The increase in INaP density was accompanied with changes in activation indicating that in the adult group a significant INaP fraction becomes available at more negative membrane potentials than in the young group, which increases the contribution of INaP in amplification of low depolarizing inputs. 5. Conclusions In summary, taking into account functional significance of INaP in physiological and pathological conditions increase in the density and hyperpolarizing shift of activation of persistent Na+ current in CA1 hippocampal neurons of adult rats may have a substantial influence on the cellular excitation promoting hyperexcitability in this region of adult brain. Acknowledgements This work was supported by the State Foundation of Fundamental Research of Ukraine F46.2/001, State Agency on Science, Innovations and Information of Ukraine and NIH grant 1R03TW008228-01A1. References [1] W.E. Crill, Persistent sodium current in mammalian central neurons, Annu. Rev. Physiol. 58 (1996) 349–362. [2] M.D. Baker, H. Bostock, Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture, J. Neurophysiol. 77 (1997) 1503–1513. [3] N. Agrawal, B.N. Hamam, J. Magistretti, A. Alonso, D.S. Ragsdale, Persistent sodium channel activity mediates subthreshold membrane potential oscillations and low-threshold spikes in rat entorhinal cortex layer V neurons, Neuroscience 102 (2001) 53–64. [4] Y. Amitai, Membrane potential oscillations underlying firing patterns in neocortical neurons, Neuroscience 63 (1994) 151–161.
O. Lunko et al. / Neuroscience Letters 559 (2014) 30–33 [5] A. Alonso, R.R. Llinas, Subthreshold Na+ -dependent theta-like rhythmicity in stellate cells of entorhinal cortex layer II, Nature 342 (1989) 175–177. [6] P.C. Schwindt, W.E. Crill, Amplification of synaptic current by persistent sodium conductance in apical dendrite of neocortical neurons, J. Neurophysiol. 74 (1995) 2220–2224. [7] M.T. Do, B.P. Bean, Subthreshold sodium currents and pacemaking of subthalamic neurons: modulation by slow inactivation, Neuron 39 (2003) 109–120. [8] S. Tazerart, L. Vinay, F. Brocard, The persistent sodium current generates pacemaker activities in the central pattern generator for locomotion and regulates the locomotor rhythm, J. Neurosci. 28 (2008) 8577–8589. [9] M. Vreugdenhil, G. Hoogland, C.W. van Veelen, W.J. Wadman, Persistent sodium current in subicular neurons isolated from patients with temporal lobe epilepsy, Eur. J. Neurosci. 19 (2004) 2769–2778. [10] S. Chen, H. Su, C. Yue, S. Remy, M. Royeck, D. Sochivko, T. Opitz, H. Beck, Y. Yaari, An increase in persistent sodium current contributes to intrinsic neuronal bursting after status epilepticus, J. Neurophysiol. 105 (2011) 117–129. [11] E.M. Horn, T.G. Waldrop, Hypoxic augmentation of fast-inactivating and persistent sodium currents in rat caudal hypothalamic neurons, J. Neurophysiol. 84 (2000) 2572–2581.
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[12] C. Alzheimer, P.C. Schwindt, W.E. Crill, Postnatal development of a persistent Na+ current in pyramidal neurons from rat sensorimotor cortex, J. Neurophysiol. 69 (1993) 290–292. [13] E. Isaeva, A. Hernan, D. Isaev, G.L. Holmes, Thrombin facilitates seizures through activation of persistent sodium current, Ann. Neurol. 72 (2012) 192–198. [14] D. Isaev, G. Ivanchick, V. Khmyz, E. Isaeva, A. Savrasova, O. Krishtal, G.L. Holmes, O. Maximyuk, Surface charge impact in low-magnesium model of seizure in rat hippocampus, J. Neurophysiol. 107 (2012) 417–423. [15] C.R. French, P. Sah, K.J. Buckett, P.W. Gage, A voltage-dependent persistent sodium current in mammalian hippocampal neurons, J. Gen. Physiol. 95 (1990) 1139–1157. [16] T.K. Aman, T.M. Grieco-Calub, C. Chen, R. Rusconi, E.A. Slat, L.L. Isom, I.M. Raman, Regulation of persistent Na current by interactions between beta subunits of voltage-gated Na channels, J. Neurosci. 29 (2009) 2027–2042. [17] J.Y. Ma, W.A. Catterall, T. Scheuer, Persistent sodium currents through brain sodium channels induced by G protein betagamma subunits, Neuron 19 (1997) 443–452.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Persistent sodium current properties in hippocampal CA1 pyramidal neurons of young and adult rats Oleksii Lunko a , Dmytro Isaev a,b , Oleksandr Maximyuk a,b , Gleb Ivanchick a , Vadym Sydorenko a , Oleg Krishtal a,b , Elena Isaeva a,∗ a b
Department of Cellular Membranology, Bogomoletz Institute of Physiology, Kiev 01024, Ukraine State Key Laboratory for Molecular and Cellular Biology, Kiev 01024, Ukraine
h i g h l i g h t s • • • • •
Persistent sodium current is an important contributor to cellular and network excitability. We analyzed changes in INaP properties during maturation. CA1 pyramidal cells were isolated from hippocampus of P12-16 and P60-75 rats. INaP density is substantially increased in the adult group. Hyperpolarization shift of INaP activation with increasing postnatal age.
a r t i c l e
i n f o
Article history: Received 12 July 2013 Received in revised form 14 November 2013 Accepted 18 November 2013 Keywords: Persistent tetrodotoxin-sensitive sodium current Hippocampal CA1 pyramidal neurons Postnatal development
a b s t r a c t Persistent tetrodotoxin-sensitive sodium current (INaP ) plays an important role in cellular and neuronal network excitability in physiological conditions and under different pathological circumstances. However, developmental changes in INaP properties remain largely unclear. In the present study using whole cell patch clamp technique we evaluated INaP properties in CA1 hippocampal pyramidal neurons isolated from young (postnatal day (P) 12–16) and adult (P60-75) rats. We show that the INaP density is substantially larger in the adult group. Although INaP inactivation characteristics were found to be similar in both groups, voltage dependence of INaP activation is shifted to more negative membrane potentials (young: −48.6 ± 0.5 mV vs. adult: −52.4 ± 0.2 mV, p < 0.01). Our data indicates the increase of INaP contribution in the basal membrane sodium conductivity in the mature hippocampus. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Slowly inactivating “persistent” Na+ current (INaP ) mediated by sodium channels with activation near the resting membrane potential was found in various central and peripheral neurons including neocortex, entorhinal cortex, thalamus, cerebellum, hippocampus and dorsal root ganglia (see [1,2] for ref). INaP plays an important role in subthreshold oscillations of membrane potential and sustains recurrent firing in response to membrane depolarization [3–5]. Moreover, INaP can amplify synaptic potentials and contributes to intrinsic pacemaking activity of different neurons [6–8]. Several reports indicate that pathological conditions, such as chronic epilepsy and hypoxia, can be accompanied by
∗ Corresponding author at: Department of Cellular Membranology, Bogomoletz Institute of Physiology, Bogomoletz str. 4, Kiev 01024, Ukraine. Tel.: +38 044 256 2519; fax: +38 044 256 2000. E-mail address: [email protected] (E. Isaeva). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.035
enhancement of INaP conductance implying involvement of this current in pathological synchronization [9–11]. Despite the significant role of INaP in the neuronal function little is known about age dependent changes in its properties. During last three decades after discovering of INaP only few studies were devoted to investigation of developmental changes in the expression of this current. Alzheimer with colleagues [12] showed threefold increase in the INaP density during first three postnatal weeks in pyramidal neurons of sensorimotor cortex. Authors suggested that INaP plays an important role in the control of excitability during early postnatal development. This data is in agreement with our previous report where activation of INaP substantially facilitated seizure-like activity in immature hippocampus [13]. However, developmental changes in INaP properties through the late adolescent period remain unclear. In the present study, patch clamp recordings were made from hippocampal CA1 pyramidal cells isolated from rats at the second postnatal week (postnatal days (P) 12–16) and at P60-75. Here we demonstrate substantial changes in both current density and steady-state activation of INaP during maturation.
O. Lunko et al. / Neuroscience Letters 559 (2014) 30–33
2. Materials and methods All experimental procedures were performed in accordance with the guidelines set by the National Institutes of Health for the humane treatment of animals and approved by the Animal Care Committee of Bogomoletz Institute of Physiology. 2.1. Slices and acutely isolated neurons preparations The hippocampal slice preparation and neuron dissociation procedure was performed as described previously with some modifications [14]. Briefly, young (P12-16, N = 19) and adult (P60-75, N = 23) Wistar rats were anesthetized with sevoflurane, decapitated and brain was rapidly moved into ice-cold oxygenated (95% O2 , 5% CO2 ) dissecting solution contained (in mM): 130 NaCl, 5 KCl, 0.1 CaCl2 , 5 MgCl2 , 1 NaH2 PO4 , 1 Na2 HPO4 , 26 NaHCO3 , 10 glucose. Hippocampal slices (400–500 m) were cut using a vibratome (MA752, Campden Instrument, Loughborough, UK) and kept for at least an hour at room temperature (22–24 ◦ C) in incubation solution contained (in mM): 130 NaCl, 5 KCl, 2 CaCl2 , 2 MgCl2 , 1 NaH2 PO4 , 1 Na2 HPO4 , 26 NaHCO3 , 10 glucose. Slices were then transferred to sucrose-based solution contained (in mM): 290 sucrose, 3 KCl, 0.5 CaCl2 , 2 MgCl2 , 10 HEPES, 15 glucose, 2 mg/ml pronase E (32 ◦ C). After 15 min of enzymatic treatment slices were rinsed with artificial cerebrospinal fluid (ACSF) contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2 , 2 MgCl2 , 20 HEPES (pH adjusted to 7.4 by NaOH). Isolation of neurons from pyramidal CA1 region of hippocampus was performed using vibrodissociation technique [14].
31
Obtained data were summarized and fitted with mono exponential function. 2.3. Data analysis Statistical data analysis was performed using Prism 5 (GraphPad, La Jolla, CA) and Origin 7.5 (OriginLab, Northampton, MA) software. The Shapiro–Wilk test was used to estimate normality of the data for each group. Statistical analysis of the development changes in INaP amplitude and density was performed using unpaired twosample Student’s t-test. Extra sum-of-squares F-test was used for voltage dependence of INaP conductance fit comparison. All data in the text were presented as the mean ± SE. 2.4. Chemicals TTX was obtained from Tocris (Ellisville, MO). All other chemicals were purchased from Sigma (St. Louis, MO). 3. Results Whole cell INaP were elicited in isolated hippocampal CA1 pyramidal cells of young (P12-16) and adult (60–75) rats using slow ramp depolarization from holding potential −80 mV to 0 mV (30 mV/s) [13]. Fig. 1A shows five superimposed responses evoked
2.2. Electrophysiological recordings Conventional patch clamp technique in the whole cell configuration was used for the recording of INaP in acutely isolated pyramidal neurons. Extracellular solution consisted (in mM): 130 NaCl, 2 CaCl2 , 1.3 MgCl2 , 20 TEACl, 10 HEPES and 0.4 CdCl2 (pH was adjusted to 7.35 with NaOH). Patch pipettes were filled with an intracellular solution contained (in mM): 120 CsF, 5 NaCl, 30 TEACl, 10 EGTA, 10 Tris-HCl (pH was adjusted to 7.2 with CsOH). Pipette resistances were ranged from 3 to 5 M. All recordings were made using model 2400 patch clamp amplifier (A-M Systems, Carlsborg, WA). Recordings were digitized at 2 kHz by analogue-to-digital converter (NI PCI-6221, National instruments, Austin, TX, USA) and filtered offline for further analysis. Liquid junction potential was determined and command voltages were corrected accordingly. All experiments were made at the room temperature. INaP was elicited by slow depolarizing ramp voltage protocol (from −80 mV to 0 mV, 30 mV/s). Current–voltage relationships of INaP were obtained by subtraction of five averaged recordings before and after treatment with 1 M tetrodotoxin (TTX). The capacitance of cells was estimated from the change in membrane charge, determined from the integrated capacity transients evoked by a 10 mV hyperpolarizing step from a holding potential (Vh ) of −80 mV. Voltage dependence of INaP conductance (G) was calculated from I/(V − Vrev ), where I is the amplitude of INaP during the test depolarization (V), and Vrev is the Na+ reversal potential calculated from the Nernst equation for the given extra- and intracellular Na+ concentrations (82 mV throughout). To evaluate the voltage-dependence of INaP steadystate inactivation the conditioning prepulses varied from −80 to −10 mV (10 s duration, 10 mV step) were preceded each standard voltage ramp stimulation to elicit INaP . All voltage-dependence relationships were normalized to the maximal conductance for each recording, then summarized for each group and fitted with a Boltzmann function. The time-dependence of inactivation was evaluated by applying conditioning prepulses to −10 mV at various duration (from 100 ms to 10 s) before a voltage ramp protocol eliciting INaP .
Fig. 1. Persistent tetrodotoxin-sensitive Na+ current in hippocampal CA1 pyramidal neurons. (A) Representative averaged currents of five superimposed traces evoked by ramp voltage depolarization from −80 mV to 0 mV before and after exposure to 1 M TTX. (B) Examples of INaP measured in hippocampal CA1 pyramidal neurons isolated from P12 (1) and P67 (2) rats (current traces were obtained by subtraction of TTX resistant current from total current).
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O. Lunko et al. / Neuroscience Letters 559 (2014) 30–33
−52.4 ± 0.2 mV (n = 26) in adult group, p < 0.01). Next we examined inactivation properties of INaP for both ages. Fig. 2B,C demonstrates that the voltage dependence of INaP inactivation (V1/2 : −43.5 ± 2.4 mV (n = 5) in young vs. −43.2 ± 2.7 mV (n = 6) in adult group) as well as INaP inactivation onset ( decay : 3.1 ± 0.3 s in young (n = 7) vs. 2.9 ± 0.4 s (n = 8) in adult group) is similar for both groups suggesting that the ageing does not affect inactivation properties of INaP . 4. Discussion
Fig. 2. Effect of age on the gating properties of persistent Na+ current. (A) Averaged steady-state activation curves (G/Gmax ) were fitted by Boltzmann equation for both young (white) and adult (grey) rats. The activation curve in the adult group was shifted by 3.8 ± 0.7 mV to more negative membrane potentials as compared to the young group. (B) Summarized data for voltage-dependent inactivation revealed no changes in the steady-state inactivation of INaP between young and adult rats. (C) Time dependence of INaP inactivation was found to be virtually the same for both ages. Stimulation protocols for (B) and (C) are shown in the corresponding insets.
by the ramp protocol in the absence and presence of 1 M TTX recorded from CA1 pyramidal cell of P12 rat. Subtraction of corresponding TTX data from each recording resulted in IV curves of INaP (Fig. 1B). INaP was activated at near −70 mV and reached its peak between −40 mV and −30 mV. The peak INaP amplitude was substantially higher in the adult group (adult: 65.5 ± 6.8 pA (n = 26); young: 35.4 ± 4.7 pA (n = 16), p < 0.01). The likely cause of such changes could be an increase in the INaP current density in adult animals. Indeed, the INaP density expressed as the ratio of the peak INaP amplitude to cell capacitance was significantly higher in the adult group (adult: 3.1 ± 0.2 pA/pF (n = 26) vs young: 1.8 ± 0.2 pA/pF (n = 16); p < 0.01). The cell capacitance was found to be virtually the same for both groups (young: 20.0 ± 1.1 pF; adult: 20.5 ± 1.4 pF, p = 0.8). Fig. 2A illustrates developmental changes in the steady-state activation of INaP . While the slope factor was unchanged (young: 5.6 ± 0.4 (n = 16) vs in adult: 5.5 ± 0.1 (n = 26), p = 0.7), the steadystate current activation curve in the adult group was shifted by 3.8 ± 0.7 mV to more negative membrane potentials as compared to the young group (−48.6 ± 0.5 mV in young (n = 16) vs.
The main findings of the present study are significant increase in the INaP density and hyperpolarization shift of activation of Na+ channel responsible for INaP in CA1 hippocampal neurons during maturation. Thus, contribution of persistent Na+ conductance in this region is larger in mature brain: INaP amplitude is increased and this current becomes manifested at lower depolarization voltages. It was shown that in hippocampal CA1 pyramidal cells most of Na+ channels responsible for INaP located at or near the somata [15]. To preserve natural cell maturation in the present study we used isolated neurons consisted somata with small dendritic arborizations. So we could not exclude a possibility that a fraction of dendritic Na+ channels contributing to INaP could have different course of maturation. The increase in INaP amplitude during postnatal development cannot be attributed to changes in the membrane capacitance or in inactivation characteristics since these parameters were similar for both age groups. However, the density of INaP was considerably increased in the adult group. This increase in the INaP conductance could be attributed to a number of mechanisms, including increase in the expression or changes in a subunit composition of Na+ channel, which could lead to changes in channel properties, the increase or decrease in the influence of endogenous factors which can also modulate INaP properties [13,16,17]. The increase in INaP density was accompanied with changes in activation indicating that in the adult group a significant INaP fraction becomes available at more negative membrane potentials than in the young group, which increases the contribution of INaP in amplification of low depolarizing inputs. 5. Conclusions In summary, taking into account functional significance of INaP in physiological and pathological conditions increase in the density and hyperpolarizing shift of activation of persistent Na+ current in CA1 hippocampal neurons of adult rats may have a substantial influence on the cellular excitation promoting hyperexcitability in this region of adult brain. Acknowledgements This work was supported by the State Foundation of Fundamental Research of Ukraine F46.2/001, State Agency on Science, Innovations and Information of Ukraine and NIH grant 1R03TW008228-01A1. References [1] W.E. Crill, Persistent sodium current in mammalian central neurons, Annu. Rev. Physiol. 58 (1996) 349–362. [2] M.D. Baker, H. Bostock, Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture, J. Neurophysiol. 77 (1997) 1503–1513. [3] N. Agrawal, B.N. Hamam, J. Magistretti, A. Alonso, D.S. Ragsdale, Persistent sodium channel activity mediates subthreshold membrane potential oscillations and low-threshold spikes in rat entorhinal cortex layer V neurons, Neuroscience 102 (2001) 53–64. [4] Y. Amitai, Membrane potential oscillations underlying firing patterns in neocortical neurons, Neuroscience 63 (1994) 151–161.
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