HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) A corrigendum has been published All Versions of this Article: 90/3/1635    most recent 00150.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (19) Citing Articles via Google Scholar Google Scholar Articles by Rybak, I. A. Articles by McCrimmon, D. R. Search for Related Content PubMed PubMed Citation Articles by Rybak, I. A. Articles by McCrimmon, D. R.

Sodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat

¹ School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104; ² Department of Physiology and Institute for Neuroscience, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611-3008

Submitted 18 February 2003; accepted in final form 15 May 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Rapidly inactivating and persistent sodium currents have been characterized in acutely dissociated neurons from the area of rostroventrolateral medulla that included the pre-Bötzinger Complex. As demonstrated in many studies in vitro, this area can generate endogenous rhythmic bursting activity. Experiments were performed on neonate and young rats (P1-15). Neurons were investigated using the whole cell voltage-clamp technique. Standard activation and inactivation protocols were used to characterize the steady-state and kinetic properties of the rapidly inactivating sodium current. Slow depolarizing ramp protocols were used to characterize the noninactivating sodium current. The "window" component of the rapidly inactivating sodium current was calculated using mathematical modeling. The persistent sodium current was revealed by subtraction of the window current from the total noninactivating sodium current. Our results provide evidence of the presence of persistent sodium currents in neurons of the rat rostroventrolateral medulla and determine voltage-gated characteristics of activation and inactivation of rapidly inactivating and persistent sodium channels in these neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The persistent sodium current (I_NaP) has been found important for intrinsic pacemaker activity in many types of neurons (e.g., see Alonso and Llinàs 1989; Bevan and Wilson 1999; Dickson et al. 1997; Llinàs et al. 1991; Pape and Driesang 1998; Pennartz et al. 1997; Takakusaki and Kitai 1997), yet usually represents 1–4% of the total sodium current (Crill 1996). This current is expressed together with the "classical," rapidly inactivating (or fast) sodium current (I_Naf), has an activation threshold 10 mV more negative than activation threshold of I_Naf and is characterized by a sustained long-lasting activation during depolarization (Crill 1996).

It has been suggested that I_NaP plays an essential role in the activation of pacemaker neurons in the pre-Bötzinger Complex (pBC), a region within the rostroventrolateral medulla (RVLM), in vitro (Butera et al. 1999; Del Negro et al. 2001; Smith et al. 2000). Del Negro et al. (2002) experimentally characterized I_NaP in pBC neurons. This study, however, focused exclusively on a noninactivating component of sodium current. The authors did not characterize I_Naf and hence did not separate I_NaP from the total noninactivating sodium current. The objective of our study was to experimentally characterize voltage-gated and kinetic properties of both I_Naf and I_NaP in the area of RVLM including the pBC. A preliminary report of this work was published in abstract form (Shevtsova et al. 2002).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Acutely dissociation procedure

Neurons were dissociated according to techniques described by McCrimmon et al. (2001). Briefly, newborn and young (1–15 days old) Sprague-Dawley rats were anaesthetized using isoflurane and decapitated. Sagittal Vibratome (Series 1000, Vibratome, St Louis, MO) slices of the brain stem (300-µm-thick) were collected in Tyrode's solution containing (in mM) 150 NaCl, 4 KCl, 2 CaCl₂, 2 MgCl₂, 10 HEPES, and 10 glucose, pH 7.4, 300 ± 5 mosM/l. The slices were incubated for 20 min at 33°C in oxygenated minimum essential medium (MEM; Life Technologies, Grand Island, NY) with (in mM) 10 HEPES, 5 L-cysteine and 0.5 EDTA, pH 7.2 to which 20 U/ml of papain (Worthington, Lakewood, NJ) was added. After enzymatic digestion, slices were washed three times in MEM-HEPES to which 1 mg/ml trypsin inhibitor and 1 mg/ml bovine serum albumin had been added.

Our intention was to study neurons from the region of RVLM that included the area of suggested location of the pBC. This area was defined in the sagittal slices by its characteristic distances from the fluorescent-labeled facial and compact/semicompact ambiguous nuclei. These cranial motoneurons were labeled by subcutaneous injection of Fluoro-Gold (50 mg/kg; Fluorochrome, Denver, CO) after birth (Ptak et al. 2001). While viewing the slices at low magnifications with an inverted microscope and epifluorescent ultraviolet illumination, a small region of the RVLM around the suggested location of the pBC was dissected from the slice and then dissociated mechanically using a series of fire-polished Pasteur pipettes. The cell suspension was centrifuged at 1,000 rpm for 10 min, then resuspended, plated onto 35-mm Petri dishes and placed on the stage of an inverted microscope.

Electrophysiological recordings

Borosilicate pipettes (WPI) were pulled, coated with Silicon elastomer (Sylgard, Dow Corning) and fire-polished. Electrode resistance was 1.5–4 M when filled with internal solution containing (in mM) 130 N-methyl-D-glucamine, 10 EGTA, 20 HEPES, 20 CsCl, 2 MgCl₂, 12 phosphocreatine, 2 Mg-ATP, 0.7 Na₂GTP, and 0.1 leupeptin, pH 7.2 with CsOH/H₂SO₄ (265 ± 5 mosM/l). The rapidly inactivating sodium current was recorded using an external solution consisting of (in mM) 15 NaCl, 110 TEA-Cl, 10 HEPES, 10 CsCl, 1 MgCl₂, 2 BaCl₂, and 0.3 CdCl₂ buffered to pH 7.4 with CsOH (300 ± 5 mosM/l). For measurement of persistent sodium current, the external solution contained (in mM) 115 NaCl, 45 TEA-Cl, 10 CsCl, 10 HEPES, 1 MgCl₂, 2 BaCl₂, and 0.3 CdCl₂ buffered to pH 7.4 with CsOH (300 ± 5 mosM/l). To avoid enhancing effect of low extracellular [Ca²⁺] on the persistent sodium current (Su et al. 2001), Ca²⁺ was replaced by the same concentration of Ba²⁺ (Magistretti and Alonso 1999). Leak current was revealed by blocking sodium currents with 300 nM TTX and then subtracted. All reagents were obtained from Sigma (St. Louis, MO).

Data acquisition and analysis

Voltage-clamp recordings were made at room temperature (21–22°C) with an Axopatch 200A amplifier (Axon Instruments). After gigaohm seal formation and cell rupture, series resistance was compensated (80–90%) and monitored periodically. Data were acquired with PCLAMP-6 software, CLAMPEX and CLAMPFIT (Axon Instrument), and analyzed with IgorPro (WaveMetrics).

To characterize steady-state properties of I_Naf, the standard activation and inactivation protocols were used (Hille 2001). To investigate the steady-state activation, currents were elicited with step depolarization from a holding potential of –80 mV to the potentials between –65 and +20 mV (using 5-mV steps). The conductance values for I_Naf were calculated by applying the extended Ohm's law in the form: g_Naf = I_Naf/(V – E_Na), where E_Na is the reversal (Nernst) Na⁺ potential calculated for the solution used in this protocol, and V is the step potential. Data were normalized and fit to the third-order Boltzmann function , where m_Naf is the steady state of activation variable, V_1/2mNaf is the half activation voltage, k_mNaf is the slope factor.

To investigate the steady-state inactivation, currents elicited by a step depolarization to 20 mV from holding potentials between –115 and –20 mV (100-ms prepulse; 5-mV step). The steady-state inactivation curve was estimated by way of normalizing peak current, plotting the normalized currents versus potentials, and fitting the resultant curve to the first-order Boltzmann function I/I_max = h_Naf = {1 + exp[(V – V_1/2hNaf)/k_hNaf]}^–1, where _hNaf is the steady state of inactivation variable, V_1/2hNaf is the half inactivation voltage, k_hNaf is the slope factor.

The activation time constant, _mNaf(V), was calculated from the time-to-peak [t_o(V)] data in each voltage step (V) during the activation protocol by fitting to first-order exponentials in the form: I(t, V) = I_max(V) · {1 – exp[t/_mNaf(V)]}/{1 – exp[t_o(V)/_mNaf(V)]}. To characterize the voltage dependence of the activation time constant, we accepted a formal description in which the maximum value of the time constant corresponded to the half activation voltage (e.g., see Butera et al. 1999). Hence _mNaf(V) was fit to the function , where V_1/2mNaf is the half activation voltage, k_mNaf is the activation time constant slope factor, and is the maximal value of activation time constant at V = V_1/2mNaf.

The inactivation time constant, _hNaf(V), was evaluated from the data on current decay for each voltage step during the activation protocol by fitting to first-order exponentials in the form: I(t, V) = [I_i(V) – I_SS(V)] · exp[–(t – t_i)/_hNaf(V)] + I_SS(V), t t_i, where I_i(V) is the current value close to I_max(V) at the moment t_i, and I_SS(V) is the minimal (steady state) value of the decayed current. Similar to the activation time constant, the voltage dependence of inactivation time constant was fit to the function , where V_1/2hNaf is the half inactivation voltage, k_hNaf is the inactivation time constant slope factor, and is the maximal value of inactivation time constant at V = V_1/2hNaf.

The experimentally characterized parameters for activation and inactivation of I_Naf, (V_1/2mNaf, k_mNaf, , k_mNaf, V_1/2hNaf, k_hNaf, , _khNaf) were then incorporated in a computer model (see following text), which was used for simulation of the experimental activation protocol. The maximal conductance _Naf was found in computer simulation to match the value of maximal sodium current (I_max) in the model to that in the experimental protocol.

To experimentally characterize biophysical properties and voltage-gated characteristics of the I_NaP, a slow depolarizing ramp increase of voltage under voltage-clamp conditions is usually applied (e.g., see Magistretti and Alonso 1999; Parri and Crunelli 1998; Taddese and Bean 2002). It is considered that the rapidly inactivating component of sodium current (I_Naf) is eliminated under these experimental conditions. However, the problem persists because I_Naf has a noninactivating or "window" component resulting from the overlap of its voltage-dependent activation and inactivation characteristics (Baker and Bostock 1997; French et al. 1990; Magistretti and Alonso 1999). The window component (I_NafW) is comparable with the I_NaP in both the peak magnitude and activation threshold. Similar to the I_NaP, the I_NafW persists when voltage is slowly increasing. Therefore, the voltage-gated characteristics of I_NaP, if obtained without accounting the window current, are likely to characterize a mixture of persistent and window currents. Hence, to characterize a "pure" I_NaP, it is necessary to study both I_Naf and I_NaP in the same neuron and to find a method allowing for separation of the I_NaP component from the total noninactivating sodium current. In the present study, we have made an attempt to characterize both fast and persistent sodium currents using a combination of experimental studies and computer modeling.

A slow depolarizing voltage ramp protocol was used to promote inactivation of the rapidly inactivating component of sodium current. The voltage was increased from –80 to 20 mV (at ramps of 100 and 75 mV/s). The evoked current (I_NaTot) was considered to consist of the persistent sodium and window currents (I_NaTot = I_NaP + I_NafW). The computer model was used to simulate the slow ramp protocol and find the voltage dependence of the window current I_NafW. The I_NaP was obtained by subtracting the simulated window current from the experimentally measured total current (I_NaP = I_NaTot – I_NafW). The conductance value for persistent sodium current was calculated from I_NaP by applying the extended Ohm's law in the form: g_NaP = I_NaP/(V – E_Na), where E_Na is the reversal Na⁺ potential calculated for the solution used in this protocol. Data were normalized and fit to the first-order Boltzmann function g_NaP/_NaP = m_NaP = {1 + exp[–(V – V_1/2mNaP)/k_mNaP]}^–1, where m_NaP is the steady state of activation variable, V_1/2mNaP is the half activation voltage, k_mNaP is the slope factor, and _NaP is the maximal conductance of the persistent sodium current.

All average values were expressed as mean ± SD.

Mathematical and computer modeling

The I_Naf was described according to the classical Hodgkin-Huxley formalism

(1)

Activation and inactivation were described by the following differential equations

(2)

where the steady-state activation and inactivation were described as

(3)

and the voltage-dependent time constants for activation and inactivation were

(4)

The I_NaP had no inactivation and was described as follows

(5)

All simulations were performed on a Pentium IV computer, 1.7 GHz/512 MB (DELL) with a Windows 2000 operating system using MATLAB 6.0 (MathWorks) ode15s routine for solution of differential equations.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Characterization of the rapidly inactivating sodium current

Figure 1, A and B illustrates sodium currents in a representative neuron evoked by standard activation and inactivation protocols respectively. Steady-state plots (see Fig. 1C) were constructed for this neuron following the subtraction of the TTX-insensitive leak current. Activation data were re-calculated for normalized conductance and fit to the third-order Boltzmann function m³_Naf, where m_Naf was defined by Eq. 3 (see METHODS). The first-order Boltzmann function h_Naf (see Eq. 3 in METHODS) was used to fit the inactivation data (Fig. 1C).

View larger version (41K): [in this window] [in a new window]   FIG. 1. Activation and inactivation characteristics of rapidly inactivating sodium current. A and B: current traces in a representative neuron (TTX-subtracted) evoked by protocols used to study the voltage dependence of activation (A) and inactivation (B). C: plots of the voltage dependence of steady-state activation () and inactivation (). The activation data for this neuron fits the 3rd-order Boltzmann function where mNaf is defined by Eq. 3 (see METHODS) with the half activation voltage V1/2mNaf = –45.6 mV and the slope factor kmNaf = 6.9 mV. The inactivation data fits the first-order Boltzmann function hNaf (see Eq. 3 in METHODS) with the half inactivation voltage V1/2hNaf = –68.4 mV and the slope factor khNaf = 10.1 mV. D1 and D2: plots of the time constants for activation () in D1) and inactivation () in D2) of the same neuron fit to Eq. 4, with the maximal value of activation time constant ms and kmNaf = 12.0 mV (D1) and the maximal value of inactivation time constant ms and khNaf = 12.7 mV (D2). E: traces of rapidly inactivating sodium current obtained in simulation of the standard activation protocol. F: overlay of the traces for the rapidly inactivating sodium current obtained in simulation at Naf = 73 nS and the corresponding experimental traces. The magnitude of Naf = 73 was found in simulation to fit the maximal current produced by the voltage step from –80 to –25 mV to the maximal current in the experimental trace corresponding to the same voltage step (pointed by arrow). Overlay of simulated and experimental traces for 3 other steps (from –80 to –15, –35, and –45 mV) are shown as examples.

Nine dissociated neurons were characterized. The average half activation voltage V_1/2mNaf = –43.8 ± 2.3 mV and the slope factor k_mNaf = 6.0 ± 0.8 mV. The average half inactivation voltage V_1/2hNaf = –67.5 ± 3.6 mV and the slope factor k_hNaf = 10.8 ± 2.4 mV. Figure 2A shows steady-state activation (fit to the 3rd-order Boltzmann function) and inactivation (fit to the 1st-order Boltzmann function) curves for nine neurons studied.

View larger version (17K): [in this window] [in a new window]   FIG. 2. A: plots of the normalized steady-state activation and inactivation of the rapidly inactivating sodium conductance (fit to the 3rd-order Boltzmann function m3Naf and the first-order Boltzmann function hNaf, respectively) for 9 neurons studied (grey traces). Black traces show the normalized steady-state activation and inactivation traces corresponding to calculated average values of parameters: V1/2mNaf = –43.8 mV; kmNaf = 6.0 mV; V1/2hNaf = –67.5 mV; khNaf = 10.8 mV. B and C: plots of the time constants for activation (B) and inactivation (C) (fit to Eq. 4) for 9 neurons studied (grey traces). Black traces in B and C show the time constants for activation and inactivation corresponding to the calculated average values of parameters: ms and kmNaf = 14.0 mV (in B), and ms and khNaf = 12.8 mV (in C).

To characterize voltage dependence of time constants for fast sodium activation and inactivation, we used the suggestion that these time constants reach their maximal values at the voltages of half activation and half inactivation, respectively. The obtained experimental data were fit to the functions Eq. 4 (see METHODS). Figure 1, D1 and D2 shows examples of voltage dependences of these time constants for the representative neuron. The preceding suggestion and the functions (Eq. 4), chosen for the formal description of time constant voltage dependence, provided a good approximation of the experimental data for voltages more positive than –45 mV (R² > 0.95) (see Fig. 1, D1 and D2). At more negative voltages (V < –45 mV), the sodium current was not expressed in our experiments (see Fig. 1C). Therefore, for V < –45 mV, the type of formal description used to describe the voltage dependence of time constant was not critical for channel kinetics.

Figure 2, B and C, shows voltage dependences of, respectively, activation and inactivation time constants (fit to the functions Eq. 4) for the nine neurons studied. The average value of the maximal activation time constant ms and the slope factor k_mNaf = 14.0 ± 3.2 mV. The average value of the maximal inactivation time constant ms and k_hNaf = 12.8 ± 1.9 mV.

To define the maximal conductance of rapidly inactivating sodium current (_Naf) for each characterized neuron, we ran a computer simulation of the corresponding standard activation protocol. The model was based on Eqs. 1–4 (see METHODS). In each simulation, the parameters used were taken or drawn from the corresponding experimental measurements for each particular neuron. For example, for the representative neuron in Fig. 1, A–D, the following parameter values were used in simulation: V_1/2mNaf = –45.6 mV; k_mNaf = 6.9 mV; ms; k_mNaf = 12.0 mV; V_1/2hNaf = –68.4 mV; k_hNaf = 10.1 mV; ms; k_hNaf = 12.7 mV; E_Na = 40 mV. The initial condition for voltage was set to V(0) = –80 mV. Voltage steps were applied as follows

(6)

where Sgn(x) = {1, if x 0; 0, otherwise}.

The value of _Naf was found in computer simulation to match the value of maximal sodium current (I_max) obtained in the corresponding experimental protocol at the voltage step producing maximal current amplitude. Figure 1E shows the results of computer simulation of the standard activation protocol for the representative neuron shown in Fig. 1, A–D. For this neuron, the maximal sodium current in the model (at the voltage step from –80 to –25 mV) fit the experimentally defined maximal current of 2,255 pA (revealed at the same voltage step) at _Naf = 73 nS (see Fig. 1, E and F). In nine neurons studied, the maximal conductance for the rapidly inactivating sodium current (defined by the above method) was in the range of _Naf = 34–170 nS.

Characterization of the persistent sodium current

All of the neurons tested exhibited a non-inactivating, TTX-sensitive sodium current that was evoked by the slow (100 and 75 mV/s) ramp increase of membrane voltage from –80 to 20 mV. Figure 3 shows a noninactivating sodium current (after subtracting the TTX-insensitive component) in the representative neuron (same as in Fig. 1, A–D) obtained during the 75-mV/s ramp protocol. This current was activated at about –60 mV and reached a peak near –40 mV (Fig. 3). This total noninactivating current was presumed to consist of the persistent sodium current and the window component of the rapidly inactivating sodium currents: I_NaTot = I_NaP + I_NafW.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Non-inactivating sodium current in the representative neuron (same as in Fig. 1, A–D) obtained in the 75-mV/s ramp protocol after subtracting the TTX-insensitive component. Inset: the control trace for the total current ("control") and the TTX-insensitive current revealed by blocking the TTX-sensitive sodium current by TTX ("TTX").

The voltage-dependent window current for each tested neuron was obtained using computer simulation. The model was used to simulate experimental voltage-ramp protocols. The value of maximal conductance for the rapidly inactivating (fast) sodium current (_Naf) for each tested neuron was found in advance during simulation of the standard activation protocols. The initial condition for voltage was set to –80 mV [V(0) = –80 mV]. The voltage increased with the ramp used in the corresponding experimental protocols (v = 100 mV/s = 0.1 mV/ms or 75 mV/s = 0.075 mV/ms)

(7)

until it reached the value of V = 20 mV.

The model was also used to compare the values of parameters of voltage-dependent activation and inactivation for the and h_Naf(V, t)) and the value of the window current () obtained in simulations of the ramp voltage protocol with the steady states of these variables (). This comparison demonstrated that both rates of voltage increase used in voltage ramp protocols were sufficient to remove the rapidly inactivating component of sodium current and the remaining window current always reached its steady state. Figure 4 shows an example of this comparison for the representative neuron obtained in simulation of the 75-mV/s voltage ramp protocol.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Examples of matching the voltage-dependent activation and inactivation of the rapidly inactivating sodium conductance (A) and the window current (B), obtained in simulation of voltage ramp protocol (75 mV/s), to the steady-state traces of these variables. This comparison shows that the transient component of the rapidly inactivating sodium current is almost eliminated, and the remaining window current is close to its steady state.

The persistent sodium current (I_NaP) was calculated by subtracting the window current (I_NafW) obtained in simulation from the total sodium current (I_NaTot) obtained in the ramp voltage protocol

(8)

Figure 5A shows an example of I_NaTot and I_NafW in the representative neuron, and Fig. 5B shows the resultant I_NaP for this neuron. The conductance of the persistent sodium current was calculated from I_NaP data by applying Ohm's law: g_NaP = I_NaP/(V – E_Na). An example for the representative neuron is shown in Fig. 5C. The values of conductance were normalized and fit to the first-order Boltzmann function g_NaP/_NaP = m_NaP (see Eq. 5 in METHODS and an example in Fig. 5, D and E).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Characterization of the persistent sodium current in the representative neuron. A: superposition of the total noninactivating sodium current (75-mV/s ramp protocol) in the representative neuron (same as in Fig. 1, A–D) and the window current obtained in simulation. B: the persistent sodium current, INaP, calculated by subtraction the window current from the total noninactivating sodium current. C: the conductance of the persistent sodium current calculated from INaP by applying Ohm's law. D and E: the normalized steady-state conductance of the persistent sodium channels in the representative neuron fit to the 1st-order Boltzmann function mNaP with the half activation voltage V1/2mNaP = –44 mV and the slope factor kmNaP = 3.1 mV.

For nine neurons characterized in two voltage ramp protocols, the average half activation voltage for the persistent sodium current and the slope factor were, respectively: V_1/2mNaP = –45.6 ± 3.1 mV and k_mNaP = 3.0 ± 0.4 mV (for the 100-mV/s protocol) and V_1/2mNaP = –48.6 ± 3.5 mV and k_mNaP = 3.2 ± 0.4 mV (for the 75-mV/s protocol). Figure 6, A and B, shows steady-state activation curves (fit to the Boltzmann function) for nine neurons studied in each ramp protocol. Considering both voltage ramp protocols, the average half activation voltage for persistent sodium current in the characterized neurons V_1/2mNaP = –47.1 ± 4.1 mV and the slope factor k_mNaP = 3.1 ± 0.4 mV (Fig. 6C). The maximal conductance of the persistent sodium channels in nine neurons studied was in the range of _NaP = 0.5 – 5.5 nS.

View larger version (14K): [in this window] [in a new window]   FIG. 6. A: plots of the normalized steady-state activation of the persistent sodium conductance (fit to the 1st-order Boltzmann function mNaP) for 9 neurons studied, obtained using 100-mV/s ramp protocol (A), 75-mV/s ramp protocol (B), and both protocols (C) (grey traces). Black traces in A–C show the normalized steady-state activation corresponding to the calculated average values of parameters: V1/2mNaP = –45.6 mV and kmNaP = 3.0 mV (in A), V1/2mNaP =–48.6 mV and kmNaP = 3.2 mV (in B), and V1/2mNaP = –47.1 mV and kmNaP = 3.1 mV (in C).

To make a backward comparison between the values for I_NaP and I_NaTot obtained in simulation and those from experimental studies, we ran simulations of the corresponding ramp protocols using the parameters established for the rapidly inactivating and persistent sodium currents in each characterized neuron

(9)

Examples of matching the experimental and model curves for I_NaP and I_NaTot for the representative neuron are shown in Fig. 7, A and B.

View larger version (14K): [in this window] [in a new window]   FIG. 7. An example of the backward comparison of values for persistent, INaP (A), and total non-inactivating sodium currents, INaTot (B), obtained in simulation to those from experimental studies.

As seen in Figs. 5D and 7, A and B, the theoretical curves for both the persistent sodium conductance and the current fit well (R² > 0.95) to the corresponding experimental curves for the initial values of voltage (V < –35 mV), where I_NaP is activated. They also fit well for the values of voltage beyond the window current (V > –5 mV), where the window current is almost completely inactivated and the persistent sodium current is completely activated. This supports the correctness of our estimation of both the voltage-gated characteristics and the maximal conductance of the persistent sodium current. At the same time, there is a significant divergence between the curves within the voltage interval corresponding to inactivation of the window current (see Figs. 5D and 7, A and B). This divergence may slightly diminish the estimated parameters of channel kinetics. It could result from a number of reasons, such as overly simplistic formal description used for both rapidly inactivating and persistent sodium channels, differences in the inactivation of the rapidly inactivating sodium current between the standard and ramp experimental protocols, disregarding the maintained presence of the persistent sodium component during processing data from the standard inactivation protocol. The divergence between the experimental and theoretical curves for persistent sodium current in the range from –35 to –5 mV could also be connected with anomalous properties of I_NaP channel noise. Kay et al. (1998) studied steady-state noise characteristics of the persistent sodium conductance experimentally and found that variance of the persistent sodium current is relatively small for voltages less than –40 mV, then dramatically rises in a range from –35 to –25 mV and monotonically decreases for voltages greater than –25 mV. The maximal value of variance in their experiments was in a voltage range from –35 to –5 mV, which corresponds to the largest divergence between the experimental and theoretical curves for persistent sodium current in our study.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Some limitations of the present study

The neurons characterized in this study were taken from animals of different ages (P1–P15), and hence possible development-dependent or age-dependent sodium channel expression could influence the variability of our results. Also our studies were performed on acutely dissociated neurons, which retained little of the original axons and dendrites. Therefore characteristics obtained in this study likely relate to somatic and proximal dendritic sodium channels.

In the present study, the maximal conductance of the rapidly inactivating sodium current was determined by matching the simulated and experimental curves corresponding to one step of the standard activation protocol. Specifically, we used the step that produced maximal current in both simulation and experiment. We did not compare the entire family of curves obtained in simulation with the entire family of experimental curves. A more precise method could be based on the comparison of partial or full families of curves. This idea was not implemented in the present study, but may be used in future investigations.

The present experimental study, including its modeling component, was performed using the assumption that I_NaP and I_Naf result from the movement of sodium ions through distinct ionic channels. At the same time, it is currently unknown whether these channels are indeed separate or represent different states of uniform but kinetically more complicated sodium channels. This issue is a subject of intense debate (e.g., see Baker and Bostock 1997; Crill 1996; Parri and Crunelli 1998; Taddese and Bean 2002).

Rapidly inactivating sodium channels

This report represents the first attempt to characterize rapidly inactivating sodium currents in RVLM neurons. Analysis of the literature has shown that the steady-state characteristics of voltage-dependent activation and inactivation of I_Naf in the RVLM neurons are similar to these in neurons from other regions in the rat brain. Our data also demonstrate that rapidly inactivating sodium current in RVLM neurons has a relatively large window conductance that may produce a window component of the rapidly inactivating sodium current comparable with the persistent sodium current in the same neuron.

Persistent sodium channels

The presence of these channels in RVLM and pBC neurons has been previously reported (Del Negro et al. 2002; Koshiya et al. 2001; McCrimmon et al. 2001; Ptak et al. 2001). Koshiya et al. (2001) reported that the peak conductance of the persistent sodium channels in pBC neurons ranged from 2.4 to 4.5 nS. According to the experimental estimation of Del Negro et al. (2002), this conductance reaches a peak value of 4–5 nS. Our measurements (0.5–5.5 nS) are consistent with these data.

Comparative analysis shows that the half activation voltage for the persistent sodium current in neurons located in different brain areas is usually about or slightly more positive than –50 mV (see Table 1). Our data on the persistent sodium half activation voltage (–47.1 ± 4.1 mV) are reasonably close to this general estimation. Consequently, we assume that the voltage-gated characteristics of the persistent sodium current in the RVLM are similar to those reported for neurons from other brain areas.

Del Negro et al. (2002) reported that the half activation voltage for I_NaP in pBC neurons was about –40 mV, which significantly differs from our data for RVLM neurons and data on I_NaP in neurons from other brain areas (see Table 1). In contrast to our study, Del Negro et al. (2002) characterized sodium current without considering the contribution of the window component of rapidly inactivating sodium current that, as shown herein, is significant in RVLM neurons. This, however, cannot explain the positive shift in the half activation voltage in their data relative to our results. Also Del Negro et al. (2002) conducted functional identification of neurons before the investigation of I_NaP characteristics. They, therefore, could specifically characterize pacemaker neurons in the pBC and separate them from nonpacemaker neurons. We did not perform functional identification of characterized neurons, and hence our data relate to functionally nonidentified RVLM neurons. However, Del Negro et al. (2002) reported a lack of significant difference in voltage-gated characteristics of the I_NaP between pacemaker and nonpacemaker neurons. Therefore it is unlikely, that the difference in our data occurred because of the lack of functional identification in our studies. The other difference between the two studies was that we characterized the persistent sodium current in acutely dissociated neurons (and mostly in somatic currents), whereas Del Negro et al. (2002) characterized neurons in slices.

In summary, results of the present study provide additional evidence of the presence of the persistent sodium current in RVLM neurons. The voltage-dependent characteristics of activation and inactivation of I_NaP and I_Naf drawn from our experiments may provide a necessary basis for computational modeling and analysis of RVLM neurons.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Science Foundation Grant 0091942, National Institute of Health (NIH) NS-046062-02 and Office of Naval Research N000140210086 grants to Ilya A. Rybak, and NIH Grants HL-60097, HL-60969, and HL-072415 to Donald R. McCrimmon.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests: I. A. Rybak, School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA 19104 (E-mail: rybak{at}cbis.ece.drexel.edu).

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Alonso A and Llinàs RR. Subthreshold Na⁺-dependent theta-like rhythmicity in stellate cells of entorhinal cortex layer II. Nature 342: 175–177, 1989.[Medline]

Baker MD and Bostock H. Low-threshold, persistent sodium current in rat large dorsal root ganglion neurons in culture. J Neurophysiol 77: 1503–1513, 1997.[Abstract/Free Full Text]

Bevan MD and Wilson CJ. Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurons. J Neurosci 19: 7617–7628, 1999.[Abstract/Free Full Text]

Brown AM, Schwindt PC, and Crill WE. Different voltage dependence of transient and persistent Na⁺ currents is compatible with modal-gating hypothesis for sodium channels. J Neurophysiol 71: 2562–2565, 1994.[Abstract/Free Full Text]

Butera RJ, Rinzel JR, and Smith JC. Models of respiratory rhythm generation in the pre-Bötzinger Complex. I. Bursting pacemaker neurons. J Neurophysiol 82: 382–397, 1999.[Abstract/Free Full Text]

Crill WE. Persistent sodium current in mammalian central neurons. Annu Rev Physiol 58: 349–362, 1996.[ISI][Medline]

Del Negro CA, Johnson SM, Butera RJ, and Smith JC. Models of respiratory rhythm generation in the pre-Bötzinger Complex. III. Experimental tests of model predictions. J Neurophysiol 86: 59–74, 2001.[Abstract/Free Full Text]

Del Negro CA, Koshiya N, Butera RJ, and Smith JC. Persistent sodium current, membrane properties and bursting behavior of pre-Bötzinger complex inspiratory neurons in vitro. J Neurophysiol 88: 2242–2250, 2002.[Abstract/Free Full Text]

Dickson CT, Mena, AR, and Alonso, A. Electroresponsiveness of medial entorhinal cortex layer III neurons in vitro. Neuroscience 81: 937–950, 1997.[ISI][Medline]

French CR, Sah P, Buckett KJ, and Gage PW. A voltage-dependent persistent sodium current in mammalian hippocampal neurons. J Gen Physiol 95: 1139–1157, 1990.[Abstract/Free Full Text]

Hille B. Ion Channels of Excitable Membranes (3rd ed.). Sinauer, 2001.

Kay AR, Sigimori M, and Llinàs R. Kinetic and stochastic properties of a persistent sodium current in mature guinea pig cerebellar Purkinje cells. J Neurophysiol 80: 1167–1179, 1998.[Abstract/Free Full Text]

Koshiya N, Del Negro C, Butera RJ, and Smith JC. Persistent sodium current (INaP) in pre-Bötzinger Complex (pre-BötC) inspiratory neurons. Soc Neurosci Abstr 27: 243.3, 2001.

Llinàs RR and Alonso A. Electrophysiology of the mammillary complex in vitro. I. Tuberomammillary and lateral mammillary neurons. J Neurophysiol 68: 1307–1320, 1992.[Abstract/Free Full Text]

Llinàs RR, Grace AA, and Yarom Y. In vitro neurons in mammalian cortical layer 4 exhibit intrinsic oscillatory activity in the 10- to 50-Hz frequency range. Proc Natl Acad Sci USA 88: 897–901, 1991.[Abstract/Free Full Text]

Magistretti J and Alonso A. Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study. J Gen Physiol 114: 491–509, 1999.[Abstract/Free Full Text]

McCrimmon DR, Monnier A, Ptak K, Zimmo G, Zhang Z, and Alheid GF. Respiratory rhythm generation: PreBötzinger neuron discharge patterns and persistent sodium current. In: Frontiers in Modeling and Control of Breathing: Integration at Molecular, Cellular and Systems Levels. New York: Plenum/Kluwer Pub, 2001, p. 147–152.

Pape HC and Driesang RB. Ionic mechanisms of intrinsic oscillations in neurons of the basolateral amygdaloid complex. J Neurophysiol 79: 217–226, 1998.[Abstract/Free Full Text]

Parri HR and Crunelli V. Sodium current in rat and cat thalamocortical neurons: role of a non-inactivating component in tonic and burst firing. J Neurosci 18: 854–867, 1998.[Abstract/Free Full Text]

Pennartz CM, Bierlaagh MA, and Geurtsen AM. Cellular mechanisms underlying spontaneous firing in rat suprachiasmatic nucleus: involvement of a slowly inactivating component of sodium current. J Neurophysiol 78: 1811–1825, 1997.[Abstract/Free Full Text]

Ptak K, Alheid GF, Zhang M, Surmeier DJ, and McCrimmon DR. Characterization of persistent sodium current in the subset of neurons in pre-Bötzinger Complex neurons. Soc Neurosci Abstr 27: 633.9, 2001.

Shevtsova NA, Ptak K, McCrimmon DR, and Rybak IA. Study of sodium currents in neurons of pre-Bötzinger complex (pBC). Soc Neurosci Abstr 32: 173.5, 2002.

Smith JC, Butera RJ, Koshiya N, Del Negro C, Wilson CG, and Johnson SM. Respiratory rhythm generation in neonatal and adult mammals: the hybrid pacemaker-network model. Respir Physiol 122: 131–147, 2000.[ISI][Medline]

Su H, Alroy G, Kirson ED, and Yaari Y. Extracellular calcium modulates persistent sodium current dependent burst-firing in hippocampal pyramidal neurons. J Neurosci 21: 4173–8412, 2001.[Abstract/Free Full Text]

Taddese A and Bean BP. Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons. Neuron 33: 587–600, 2002.[ISI][Medline]

Takakusaki K and Kitai ST. Ionic mechanisms involved in the spontaneous firing of tegmental pedunculopontine nucleus neurons of the rat. Neuroscience 78: 771–794, 1997.[ISI][Medline]

This article has been cited by other articles:

				S. Wittmeier, G. Song, J. Duffin, and C.-S. Poon Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis PNAS, November 18, 2008; 105(46): 18000 - 18005. [Abstract] [Full Text] [PDF]

				I. A. Rybak, R. O'Connor, A. Ross, N. A. Shevtsova, S. C. Nuding, L. S. Segers, R. Shannon, T. E. Dick, W. L. Dunin-Barkowski, J. M. Orem, et al. Reconfiguration of the Pontomedullary Respiratory Network: A Computational Modeling Study With Coordinated In Vivo Experiments J Neurophysiol, October 1, 2008; 100(4): 1770 - 1799. [Abstract] [Full Text] [PDF]

				J. C. Smith, A. P. L. Abdala, H. Koizumi, I. A. Rybak, and J. F. R. Paton Spatial and Functional Architecture of the Mammalian Brain Stem Respiratory Network: A Hierarchy of Three Oscillatory Mechanisms J Neurophysiol, December 1, 2007; 98(6): 3370 - 3387. [Abstract] [Full Text] [PDF]

				R. W. Pace, D. D. Mackay, J. L. Feldman, and C. A. Del Negro Role of persistent sodium current in mouse preBotzinger Complex neurons and respiratory rhythm generation J. Physiol., April 15, 2007; 580(2): 485 - 496. [Abstract] [Full Text] [PDF]

				K. Ptak, G. G. Zummo, G. F. Alheid, T. Tkatch, D. J. Surmeier, and D. R. McCrimmon Sodium Currents in Medullary Neurons Isolated from the Pre-Botzinger Complex Region J. Neurosci., May 25, 2005; 25(21): 5159 - 5170. [Abstract] [Full Text] [PDF]

				J. T. Potts, I. A. Rybak, and J. F. R. Paton Respiratory Rhythm Entrainment by Somatic Afferent Stimulation J. Neurosci., February 23, 2005; 25(8): 1965 - 1978. [Abstract] [Full Text] [PDF]

				L. K. Purvis and R. J. Butera Ionic Current Model of a Hypoglossal Motoneuron J Neurophysiol, February 1, 2005; 93(2): 723 - 733. [Abstract] [Full Text] [PDF]

				C. A. Del Negro, C. Morgado-Valle, J. A. Hayes, D. D. Mackay, R. W. Pace, E. A. Crowder, and J. L. Feldman Sodium and Calcium Current-Mediated Pacemaker Neurons and Respiratory Rhythm Generation J. Neurosci., January 12, 2005; 25(2): 446 - 453. [Abstract] [Full Text] [PDF]

				E. K. Kosmidis, O. Pierrefiche, and J.-F. Vibert Respiratory-Like Rhythmic Activity Can Be Produced by an Excitatory Network of Non-Pacemaker Neuron Models J Neurophysiol, August 1, 2004; 92(2): 686 - 699. [Abstract] [Full Text] [PDF]

				N. I. Kononenko, L.-R. Shao, and F. E. Dudek Riluzole-Sensitive Slowly Inactivating Sodium Current in Rat Suprachiasmatic Nucleus Neurons J Neurophysiol, February 1, 2004; 91(2): 710 - 718. [Abstract] [Full Text] [PDF]

				J. A. Neubauer and J. Sunderram Oxygen-sensing neurons in the central nervous system J Appl Physiol, January 1, 2004; 96(1): 367 - 374. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) A corrigendum has been published All Versions of this Article: 90/3/1635    most recent 00150.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science