Effect of chronically elevated CO2 on CA1 neuronal excitability

Xiang Q. Gu,1 Jin Xue,1 and Gabriel G. Haddad1,2

1Department of Pediatrics, Section of Respiratory Medicine; and 2Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461

Submitted 4 February 2004 ; accepted in final form 26 April 2004


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To study the effect of chronically elevated CO2 on the excitability and function of neurons, we exposed mice to 7.5–8% CO2 for ~2 wk (starting at 2 days of age) and examined the properties of freshly dissociated hippocampal neurons. Neurons from control mice (CON) and from mice exposed to chronically elevated CO2 had similar resting membrane potentials and input resistances. CO2-exposed neurons, however, had a lower rheobase and a higher Na+ current density (580 ± 73 pA/pF; n = 27 neurons studied) than did CON neurons (280 ± 51 pA/pF, n = 34; P < 0.01). In addition, the conductance-voltage curve was shifted in a more negative direction in CO2-exposed than in CON neurons (midpoint of the curve was –46 ± 3 mV for CO2 exposed and –34 ± 3 mV for CON, P < 0.01), while the steady-state inactivation curve was shifted in a more positive direction in CO2-exposed than in CON neurons (midpoint of the curve was –59 ± 2 mV for CO2 exposed and –68 ± 3 mV for CON, P < 0.01). The time constant for deactivation at –100 mV was much smaller in CO2-exposed than in CON neurons (0.8 ± 0.1 ms for CO2 exposed and 1.9 ± 0.3 ms for CON, P < 0.01). Immunoblotting for Na+ channel proteins (subtypes I, II, and III) was performed on the hippocampus. Our data indicate that Na+ channel subtype I, rather than subtype II or III, was significantly increased (43%, n = 4; P < 0.05) in the hippocampi of CO2-exposed mice. We conclude that in mice exposed to elevated CO2, 1) increased neuronal excitability is due to alterations in Na+ current and Na+ channel characteristics, and 2) the upregulation of Na+ channel subtype I contributes, at least in part, to the increase in Na+ current density.

sodium ion channels; oxygen deprivation


A NUMBER OF DISEASES AND CONDITIONS are associated with tissue hypercapnia. For example, obstructive sleep apnea/hypoventilation syndrome (OSAHS) in both children and adults is associated with an elevated CO2 level. This hypercapnia also occurs in patients with various other diseases such as cystic fibrosis (5), asthma (32), and respiratory failure (22, 28). These diseases can lead to either intermittent or chronic hypercapnia. If left untreated, some of these conditions, such as OSAHS, can lead to high blood pressure and are possible risk factors for stroke (26).

The effects of chronic hypercapnia on neural properties are not clear. Although there is limited literature on the cellular and molecular mechanisms of response to high CO2 conditions, there are many unanswered questions. For example, central respiratory drive potentials recorded in artificially ventilated subjects under hyperoxic hypercapnia suggested the involvement of voltage-sensitive channels (18). In fact, many channels are reported to be CO2 sensitive, including voltage-gated connexins (27), K+ channels (29), aquaporins (31), L-type Ca2+ channels (30), and ATP-sensitive K+ channels (33). Most of these CO2 experiments, however, involved acute application of CO2. The effect of exposure to elevated CO2 for prolonged time periods is not well studied. Because the impact of hypercapnia depends on the magnitude and extent of exposure to hypercapnia, the age of the subject, and the nature of the hypercapnia (constant or intermittent), in the present work we studied mice exposed to a high CO2 level during the first 2 wk of life. We used electrophysiological and molecular methods to determine the mechanisms that were altered in response to hypercapnia. We focused our work on the hippocampus for several reasons. 1) This region is very well studied, and investigators at our laboratory (15) as well as other investigators have studied in detail the characteristics of these neurons. 2) The CA1 region demonstrates cell death in a major way during stroke or severe hypovolemic shock and hypotension, not only because of ischemia but also likely because of increased CO2. 3) It was found recently that sleep fragmentation and sleep apnea, which are associated with increased CO2 level, can affect memory, and the hippocampus is an important site for this function (12). 4) To compare the effect of CO2 with that of low O2 in mice, we had to use the same region that was studied in the past, i.e., hippocampal CA1.


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Chronic Exposure to Elevated CO2

A computer-controlled chamber (OxyCycler; Reming Bioinstruments, Redfield, NY) was used for the induction and maintenance of chronically elevated CO2. The chamber (Plexiglas 76.2 x 49.5 x 52.7 cm in length, width, and height, respectively) could simultaneously hold up to four mouse cages (28 x 16 x 12 cm in length, width, and height, respectively). The CO2 level was monitored and controlled by OxyCycler software at a constant concentration of 7.5–8% (Fig. 1). Oxygen in the chamber was kept at a constant concentration of 21% (data not shown) and was balanced by nitrogen. Mice at the age of 2–3 days were placed in small cages in the chamber with their dam and were exposed to CO2 for ~10–14 days, until the time of death. All CO2-exposed mice survived the period of exposure. Control mice (CON) were also placed in small cages in the same room and shared the same noise level, lighting, and environment. Pregnant mice (CD1) were purchased from Charles River Laboratories (Wilmington, MA).



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Fig. 1. Spontaneous activity from neurons of control mice (CON) (A) and mice exposed to chronic CO2 (B). Data were collected at the current-clamp mode with command current of 0 pA. Each trace in either A or B consists of 10 consecutive traces. Horizontal bars at left in A and B stand for the 0-mV level. Notice that in B, the membrane potential (Vm) becomes spontaneously depolarized and the neuron enters a depolarization block. Scales represent 10 mV and 25 ms.

 
Preparation of CA1 Cells

Mice at age 12–16 days were used and then killed by inhalation of isoflurane (Baxter, Deerfield, IL). Their hippocampi were quickly removed and sliced into ~10 transverse sections of 400-µm thickness. The slices were immediately transferred to a homemade container with 10 ml of fresh, oxygenated, slightly stirred HEPES buffer at room temperature. After 40 min of exposure to protease (0.1%) digestion, the slices were washed with HEPES buffer and left in the oxygenated and stirred HEPES solution. The CA1 region was then dissected out and triturated in a small (0.25 ml) volume of HEPES buffer with three pipettes of gradually reduced openings. The Albert Einstein College of Medicine Animal Care and Use Committee approved these studies.

Electrophysiological Recording and Solutions

Electrodes for whole cell recording were pulled on a Flaming/Brown micropipette puller (model P-87; Sutter Instrument, Novato, CA) from filamented borosilicate capillary glass (1.2-mm OD, 0.69-mm ID; World Precision Instruments, Sarasota, FL, or Warner Instrument, Hamden, CT). The electrodes were fire polished, and resistances were 2–5 M{Omega} for voltage-clamp experiments and 7–9 M{Omega} for current-clamp experiments in the solutions described below. Membrane potentials (Vm) and action potentials (APs) were recorded in the current-clamp mode. Input resistance (Rm) was calculated at –70 mV as 1 divided by the slope of the current trace evoked by a ramp voltage from –160 to +100 mV in the voltage-clamp mode. The slope was derived from least-squares regression analysis for 100 data points between voltage and current. Current traces in the voltage clamp were leak subtracted. Liquid junction potentials were nulled for each individual cell with the Axopatch 1C amplifier (Axon Instruments).

The HEPES-buffered solutions for the enzymatic preparation and trituration of the CA1 cells as well as the external HEPES solution used for the current-clamp experiments contained (in mM) 130 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 138 KCl, 0.2 CaCl2, 1 MgCl2, 10 HEPES (Na+ salt), and 10 EGTA, with pH adjusted to 7.4 with Tris. The external solutions for the voltage-clamp experiments contained reagents similar to those used in the current-clamp experiments, except for (in mM) 10 tetraethylammonium chloride, 5 4-aminopyridine, and 0.1 CdCl2 and the reduction of the NaCl concentration from 130 to ~117. The internal pipette solution for the voltage-clamp experiments was similar to the internal solution for the current-clamp experiments, except for the use of either CsF or CsCl instead of KCl. Investigators at our laboratory (25) previously showed that there was no difference in whole cell Na+ current recorded when CsF or CsCl was used. The osmolarity of all solutions was adjusted to 290 mosM. All recordings were performed at room temperature (22–24°C). All chemicals were purchased from Sigma (St. Louis, MO).

Recording Criteria

These criteria were previously used, as detailed in a previous publication (25). We used morphological and electrophysiological criteria.

Morphological criteria. CA1 cells were used if they had a smooth surface and a three-dimensional contour and were pyramidal in shape. Investigators at our laboratory (15, 16) and other investigators (17) have used similar criteria for freshly triturated neurons. The CA1 neurons studied were obtained from 12- to 14-day-old mice.

Electrophysiological criteria. Neurons were considered for recording if the seal resistance was >5 G{Omega}. Only neurons with a holding current of <0.1 nA (command potential –100 mV) were used in the study. Series resistance was <10 M{Omega} in neurons studied. The series resistances were compensated at the 90% level with the Axopatch 1C amplifier. Under these conditions, the error caused by uncompensated series resistances was <2.3 mV. The error was not corrected, because it was negligible. To obtain adequate voltage clamp and minimize the space-clamp problem, only neurons of small size with short processes were used in Na+ current measurements. In addition, only cells with current-voltage curves that were smoothly graded over the voltage range of activation (~–50 to –10 mV) were used, as investigators at our laboratory have done in the past (15, 16).

Immunoblotting

Hippocampal tissues from five to six animals were separately dissected out, weighed, pooled, and transferred to lysis buffer as one sample (4x vol/wt; 200 mM mannitol, 80 mM HEPES, 41 mM KOH, 1 µM pepstatin A, 1 µM leupeptin, 230 µM phenylmethylsulfonyl fluoride, and 1 mM ethylenediamine tetrahydrochloride, pH 7.5). We used a microsomal preparation according to a method described by Grassl and Aronson (14). The tissues were then homogenized with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ), and the homogenate was centrifuged at 1,000 g at 4°C for 10 min to remove cellular debris. The supernatant was recentrifuged at 100,000 g at 4°C in a Beckman SW40T rotor for 1 h. The resulting pellet was resuspended in 200–500 µl of lysis buffer, and protein concentration was determined with the use of a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). Membrane protein (30 µg) was resolved on 4–12% precast NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and electrotransferred onto polyvinylidene difluoride membranes (Immobilin-P; Millipore, Bedford, MA). Affinity-purified rabbit polyclonal antibodies against Na+ channel type I (1:600 dilution), type II (1:300), and type III (1:80) (Sigma) were applied. Protein signals were detected with the use of an enhanced chemiluminescence system (ECL; Amersham, Chalfont, UK). For normalization, all of the membranes were stripped and reprobed with affinity-purified goat polyclonal antibody to actin at 1:1,000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA).

Scanning densitometry of immunoblot films was performed on a Personal Densitometer SI scanner (Molecular Dynamics, Sunnyvale, CA) and analyzed with the aid of ImageQuant image analysis software (Molecular Dynamics). Data are presented as ratios of protein to corresponding actin. Percentage values are based on data from age-matched naive mice that were considered as 100%. Four to five pooled samples, with each sample containing five to six hippocampi from different animals, were tested for each group.

Statistical Analysis

Student's t-test was used for comparisons. All values represent means ± SE. Differences in means are considered significant if P < 0.05.


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Body, Brain, and Hippocampal Weights

We measured body, brain, and hippocampal weight in both naive and CO2-exposed mice. Neither brain weight, nor hippocampal weight, nor the ratio of hippocampus to body weight or hippocampus to brain weight was significantly different in the CO2 group compared with the naive group. However, the body weight of the CO2-treated mice (10.94 ± 0.26 g, n = 15) was modestly but significantly greater than that of the naive group (9.93 ± 0.33 g, n = 15) (P = 0.01). The ratio of brain to body weight of the CO2-treated mice was significantly smaller in CO2-exposed mice (0.0378 ± 0.001 g, n = 15) than in the naive group (0.043 ± 0.001 g, n = 15) (P = 0.004).

Neuronal Properties and Membrane Excitability

All CA1 neurons from CON mice (n = 7) and the CO2-exposed group (n = 7) fired APs when they were held at –75 mV and administered depolarizing currents in the current-clamp mode. None of the CON neurons (n = 12) and only 2 of 16 CO2-exposed neurons, however, fired spontaneous APs (Fig. 1). CA1 neurons from CO2-exposed animals had a membrane potential similar to that of CON neurons (Vm: –34 ± 1 mV, n = 16, for CO2-exposed neurons vs. –30 ± 3 mV, n = 11, for CON neurons). To determine excitability in CON and CO2-exposed neurons, we measured the rheobase. Stepping from the same Vm (–75 mV) in the current-clamp mode, the amount of current required to generate one AP was 44 ± 10 pA (n = 12) in CO2-exposed neurons, which represented only one-third that of CON neurons (113 ± 17 pA, n = 20; P = 0.002) (Fig. 2). The amplitude of evoked AP in CO2-exposed neurons was >30% greater than that of CON (data not shown), and both groups had similar input resistance.



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Fig. 2. Voltage traces with evoked action potentials (APs) in CON (A) and chronic CO2-exposed neurons (B) and mean rheobase (C). As shown in A and B, evoked APs were collected in the current-clamp mode at –75 mV with 10 depolarizing currents, starting with 10 pA and using 10-pA increments. Voltage traces in A and B start at the bottom and move upward incrementally. Scales represent 50 mV and 25 ms. In C, the minimum currents used to evoke an AP were averaged. Values are means ± SE. *P = 0.002.

 
Na+ Current Magnitude

Because there were significant differences in excitability between CO2-exposed and CON neurons, even when neurons were held from the same Vm, we examined the Na+ channel properties of both CON and CO2-exposed neurons. Steps from a holding potential of –130 to –20 mV evoked an inward current that reached a peak in <1 ms and decayed quickly to 0 current. TTX blocked that current. The average peak Na+ current in CO2-exposed neurons was 2,335 ± 364 pA (n = 28), which was almost triple the peak Na+ current in CON of 826 ± 130 pA (n = 34; P < 0.001). To eliminate the role of cell membrane surface area that contributes to the whole cell peak current, we used the current density, which was calculated as peak current divided by the area of the neuron (as reflected by whole cell capacitance measurements of 3.6 ± 0.4 pF, n = 34, in CON neurons and 4.2 ± 0.4 pF, n = 41, in CO2-exposed neurons; P = 0.12). The current density (580 ± 73 pA/pF, n = 27) in CO2-exposed neurons was more than twice that of the CON group (280 ± 51 pA/pF, n = 34; P = 0.0005).

Na+ Current Characteristics

We next examined the characteristics of the Na+ current to determine whether other differences existed that could play a role in determining the excitability of CO2-exposed and CON neurons.

Activation. The threshold for Na+ channel activation was ~–60 mV for both groups of neurons, and the Na+ current reached a peak at –20 mV for CON neurons and –30 mV for the CO2-exposed group (Fig. 3). By plotting the voltage-conductance relationship curve and fitting the relationship to the Boltzmann equation, we found that the midpoint of the curve was shifted significantly in the hyperpolarizing direction by 12 mV in the CO2-exposed group compared with that in the CON group (–34 ± 3 mV, n = 9, for CON neurons and –46 ± 3 mV, n = 7, for CO2-exposed neurons; P = 0.007). The voltage dependence of the voltage-conductance curves was not significantly different between the two groups, because the slope factors for the curves (3.4 ± 0.5 for CON and 2.6 ± 0.5 for CO2-exposed neurons) were not different.



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Fig. 3. Activation and conductance-voltage relationship of the Na+ current. As shown in A (CON) and B (CO2 exposed), current traces were collected from –70 to 0 mV with 10-mV increments from a holding potential of –130 mV for CON and chronically elevated CO2-exposed neurons. Voltage protocol is shown at bottom of A. In C, current-voltage relationship is expressed as normalized current (current at Vm divided by the peak Na+ current of the family curve, –I/Imax) at each Vm against the Vm. In D, voltage-conductance relationship is expressed as normalized conductance (g/gmax) against Vm. Curves were fitted by the Boltzmann equation.

 
Steady-state inactivation. We also studied the steady-state inactivation of the Na+ current by using a prepulse potential of 502 ms from –130 to –20 mV with an increment of 10 mV and then stepping to a command potential of –20 mV. When the ratios of each individual peak current to the maximal peak current were plotted against the prepulse potentials, the steady-state inactivation curve of CO2-exposed neurons was shifted significantly in the depolarizing direction (Fig. 4). The midpoint of this steady-state inactivation curve was –59 ± 2 mV (slope factor = 5.6 ± 0.3, n = 22) and –68 ± 3 mV (slope factor = 6.2 ± 0.4, n = 21; P = 0.004) for CO2-exposed and CON neurons, respectively, a significant difference.



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Fig. 4. Steady-state inactivation of the Na+ current. In A (CON) and B (chronic CO2), current traces were collected at –20 mV from prepulse potentials ranging from –130 to –20 mV with an increment of 10 mV and a duration of 502 ms. Voltage protocol is shown at bottom of A. Scales in A and B represent 1 nA and 10 ms. I/Imax is plotted against the prepulse potential in C. Curves were fitted by the Boltzmann equation.

 
Recovery from inactivation. When examining the recovery from inactivation, we used a two-pulse protocol with a series of intervals with an increment of 2.7 ms between the two pulses. The time constant ({tau}h) for recovery from inactivation, which could be fit by a first-order exponential equation, was significantly smaller in CO2-exposed neurons than in CON neurons (4.18 ± 0.4 ms, n = 20, and 5.8 ± 1.0 ms, n = 16, for CO2-exposed and CON neurons, respectively; P = 0.05) (Fig. 5).



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Fig. 5. Recovery from inactivation of the Na+ current. As shown in A (CON) and B (chronic CO2), 2 identical pulses were delivered with increasing intervals (t) between each pair of pulses. Voltage protocol is shown at bottom of A. Scales in A and B represent 1 nA and 10 ms. In C, recovery from inactivation of the Na+ current is plotted as the ratio of the peak of the second current to that of the first (Ipeak2/Ipeak1) against the intervals (t) in a 2-pulse voltage protocol.

 
Deactivation characteristics. We further examined the deactivation properties and the transition from the open to the resting closed state, without inactivating the channel, for both groups of neurons. We held CA1 neurons at –100 mV, depolarized them for 1 ms to –10 mV, and repolarized them to –100 mV (Fig. 6, A and B). Current traces in response to this protocol are shown separately in Fig. 6A for CON neurons and in Fig. 6B for CO2-exposed neurons, and the two traces are superimposed in Fig. 6C. The normalized current traces for CON neurons and CO2-exposed neurons are superimposed in Fig. 6D for comparison. Notice that Fig. 6C represents the actual recordings overlaid, while Fig. 6D represents scaled recordings. The averaged time constant for deactivation ({tau}d) at –100 mV, which could be fit by a first-order exponential equation, was significantly smaller for CO2-exposed neurons (0.8 ± 0.1 ms, n = 20) than for CON neurons (1.9 ± 0.3 ms, n = 19; P < 0.001) (Fig. 6E).



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Fig. 6. Deactivation of the Na+ current. Traces shown in A and B represent deactivation of the Na+ current in CON neurons (A) and neurons exposed to chronic CO2 (B). Voltage protocol is shown at bottom of A. In C, current traces from A and B are superimposed. Scale in B is applicable to A, B, and C and represents 1 nA and 10 Ms. In D, normalized current traces for both CON neurons and neurons exposed to chronic CO2 are superimposed. In E, the averaged time constants for deactivation ({tau}d) as repolarized to –100 mV are plotted for the CON and chronic CO2-exposed groups. Values are means ± SE. *P < 0.001.

 
Na+ Channel Expression

We further studied whether the increased Na+ current density is due to an increased Na+ channel expression, and, if so, which Na+ channel subtype is upregulated. Because there are three major neuronal subtypes of Na+ channels in the central nervous system (CNS), we chose specific antibodies against each neuronal isoform of Na+ channel and performed immunoblotting assays. Exposure to CO2 selectively increased the Na+ channel subtype I protein level (43%, n = 4; P < 0.05) (Fig. 7A) in the hippocampus, with no obvious increase detected for Na+ channel subtype II or III (Fig. 7, B and C). It is interesting to note that the upregulation of Na+ channel expression was less than the increase in Na+ channel current density (Fig. 8). In the hippocampi of the CO2-exposed mice, Na+ current density was elevated by 107% relative to CON, in contrast to approximately one-half of this increase in the expression of membrane Na+ channel.



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Fig. 7. Differential regulation of Na+ channel subtypes I (A), II (B), and III (C) in the 2-wk chronic CO2-exposed mice. Immunoblotting method was used to compare protein levels of Na+ channel subtypes I, II, and III between chronic CO2-exposed mice and their age-matched, wild-type controls in the hippocampus. In A–C, at top, immunoblots show representative of Na+ channel proteins and their corresponding actins; at bottom are densitometric analyses of the protein signals. In each graph, the x-axis represents experimental conditions; the y-axis depicts the relative level of Na+ channels as a ratio of Na+ channel protein to actin density per 30 µg of total membrane protein. Values are means ± SE (n = 4 for each group, except n = 5 for subtype I control). *P < 0.05. Note that Na+ channel subtype I was significantly increased (43%) in the hippocampi of mice exposed to chronic CO2 compared with CON mice.

 


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Fig. 8. Comparison between Na+ channel expression and Na+ channel current density in hippocampi of mice subjected to 2 wk of chronic CO2 exposure. Na+ channel expression was assayed by immunoblotting. Na+ current density was obtained from voltage-clamp recordings, with peak currents normalized to whole cell capacitances. Data are expressed as percent changes of control level in wild-type mice. *P < 0.05 compared with wild type. Note that elevation of Na+ channel current density is approximately twofold that of Na+ channel expression (subtypes I, II, and III) in the hippocampus.

 

    DISCUSSION
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In this work, we have shown that neurons from animals chronically exposed to elevated CO2 have higher excitability. A higher percentage (13% compared with 0% in naive neurons) of these neurons fire spontaneous APs, their rheobase was smaller (44 pA) than that of CON neurons (113 pA), and the amplitude of evoked AP in CO2-exposed neurons was 30% larger than that of CON neurons. The difference in excitability was not related to Vm, because the rheobase experiments were performed at the same voltage (–75 mV). The difference in excitability also was not related to Rm, because both CON neurons and CO2-exposed neurons had similar Rm. An important observation made in these mice is that, unlike our experiments in chronic hypoxia, these mice did not fail to grow while exposed to chronically elevated CO2. Their growth was seemingly appropriate, and no poor growth was observed, unlike our observations in mice exposed to chronic hypoxia (15).

There are at least several reasons for the increased excitability in CO2-exposed neurons. First, the Na+ current density was significantly (~110%) higher in these neurons than in CON neurons. Furthermore, the difference in Na+ current density was not a result of differences in neuronal size, because both groups had similar whole cell capacitances. This difference in Na+ current density is an important factor that could explain the difference in excitability between the two groups of neurons. Second, the activation of the Na+ channel, as predicted by the voltage-conductance curve, was much higher in CO2-exposed neurons than in CON neurons at respective voltages. For example, at –40 mV, channel-opening probability was predicted to be 27% for CON neurons but 79% for CO2-exposed neurons (Fig. 3D). Third, it seems from the steady-state inactivation curve that the Na+ channels of CO2-exposed neurons were more available for activation. For example, at –70 mV, 59% of the Na+ channels of naive neurons were available, whereas 78% were available in CO2-exposed neurons (Fig. 5C). Fourth, the time constant of recovery from inactivation was 37% smaller in CO2-exposed neurons than in CON neurons. The smaller {tau}h warrants quicker availability for another AP and shorter latency after the first AP in case of higher frequency of APs. Fifth, the time constant of deactivation at –100 mV was also much (87%) smaller in CO2-exposed than in CON neurons. The smaller {tau}d warrants quicker recovery to the resting state if there is no inactivation involved after a brief activation.

In the present work, one question that arises is related to the cause of the increase in the Na+ current density. This increase can be a result of increased channel expression, open probability, or single-channel conductance. Our immunoblotting data show that the expression of type I Na+ channel was selectively upregulated in the hippocampus, in contrast to types II and III, which were not. However, as shown in Fig. 8, this increase in the expression of membrane Na+ channel was approximately one-half of that in Na+ current density measured electrophysiologically, suggesting that increased expression was not the sole factor. The open probability and/or single-channel conductance could have been altered in the hippocampus of CO2-treated mice as well.

There are multiple subtypes of Na+ channels in the CNS (1, 4, 23, 24). These Na+ channel subtypes exhibit distinct spatial (regional and subcellular) and temporal expression patterns (2, 3, 13, 19). Our current data show that CO2 exposure increases only Na+ subtype I expression in the hippocampus. This finding is different from our previous study, in which we studied animals exposed to hypoxia. After a 2-wk exposure to intermittent hypoxia, the expression of Na+ channel subtype II was selectively reduced and not increased in the hippocampus (Zhao P, Xue J, Gu XQ, Haddad GG, and Xia Y, unpublished observation). This indicates that although both hypercapnia and hypoxia have an effect on the excitability of neurons and on Na+ channels, they actually have different effects on the expression and functional regulation of these channels.

It is important to mention that in hypoxia or hypercapnia (i.e., the present studies), CON animals were treated in the same way as the experimental animals, except for the exposure to low O2 or high CO2: they shared the same room, the same lighting, and the same noise level.

It is known that acute perfusion of CO2-HCO3 decreases the excitability of neurons in slices or acutely dissociated neurons (7–11, 16). This is the result of hyperpolarizing the resting membrane potential (10, 16) and changing the characteristics of Na+ channels (6, 16). It is not known, however, whether it is CO2 or HCO3, or both, that contributes to these effects. Recently, Bruehl and Witte (6) did an important experiment to differentiate the effects of CO2 from those of HCO3. They noticed that the midpoint of the conductance-voltage curve and of the steady-state inactivation curve shifted in the negative direction when perfusion of acutely dissociated neurons was switched to CO2-HCO3 solution. Holding HCO3 constant and changing the CO2 concentration would not have the same effects, while holding CO2 constant and varying the concentration of HCO3 had the same effects as perfusion with CO2-HCO3. Bruehl and Witte concluded that the effects of CO2-HCO3 on the excitability of the Na+ channel derived from HCO3 and not from CO2. Because they used the same pH when they switched the solutions, this eliminated the effects of extracellular pH.

Long-term effects of CO2 or HCO3 have rarely been reported (20), and these reports describe work on carotid bodies. In this report, we describe excitability in the hippocampal neurons of mice exposed to elevated CO2 levels for 14 days. The increased excitability we describe is the result of the dramatic changes in whole cell characteristics of the Na+ channel: negative shift in the midpoint of the conductance-voltage curve, positive shift in the steady-state inactivation curve, smaller {tau}h, and smaller {tau}d. We also report a larger Na+ current, a larger Na+ channel density, and a greater expression of Na+ channels in CO2-exposed neurons. The larger whole cell Na+ current that we report is in line with the findings of Bruehl and Witte (6), who reported a 35% increase in Na+ current when the external perfusing solution increased CO2-HCO3 concentration. We noted an interesting behavioral observation in these mice: they were much more active than their CON counterparts in their cages.

There is a possibility that the results we observed in our current work resulted from changes in pH. In well-controlled experiments in which CO2 and HCO3 were changed at a constant pH, there was no effect on the midpoints of the conductance-voltage curve and the steady-state inactivation curve (6). In our studies, we kept the extracellular pH and CO2 constant, and hence this cannot be a factor. However, because we did not measure intracellular pH (pHi), especially because there could be differences in membrane exchangers in two groups of neurons, pHi could be an important factor that contributed to the differences in excitability of neurons harvested from CO2-exposed and naive animals.

A number of questions can be raised from our work. For example, Why should Na+ channels be upregulated, and do they serve any beneficial function in an organism after weeks of CO2 exposure? Can this upregulation lead to seizures? How different is the response to CO2 in young, growing mice compared with older animals? How generalizable are our results regarding hippocampal neurons to other groups of cells in the CNS? These and other questions cannot be answered easily on the basis of our current work. However, we think that while an abrupt increase in CO2 could lead to seizures (21), chronic exposure may not. Another important issue to consider is the resulting level of CO2 in the blood and interstitial space. While this was not measured in our animals, we suspect that PaCO2 would be ~55–60 Torr. The response to lower or higher PaCO2 may be totally different; indeed, at acute levels >90 Torr could lead to obtundation and EEG flattening, possibly because of pHi effect on Na+ channel kinetics. At this stage, we need to experimentally study different CO2 levels in mice at different developmental stages to be able to start to answer some of these questions.

In summary, we have shown in this work that young mice exposed to chronically elevated CO2 have higher excitability of hippocampal neurons. The increased excitability is due to an increase in Na+ current density and alterations in Na+ channel characteristics, including voltage-conductance curve, steady-state inactivation, recovery from inactivation, and deactivation. Furthermore, the increase in the expression of Na+ channels in CO2-exposed animals might have contributed to the increased excitability in hippocampal neurons.


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This work was supported by National Institutes of Health Grants P01 HD-32573, R01 NS-35918, and R01 HL-66327.


    ACKNOWLEDGMENTS
 
We thank Ralph Garcia, Aaron Hochberg, Hillary Sunamoto, Mary Catherine Muenker, and Orit Gavrialov for invaluable technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, Albert Einstein College of Medicine, Rose F. Kennedy Center for Research in Mental Retardation and Human Development, 1410 Pelham Parkway South, Bronx, NY 10461 (E-mail: ghaddad{at}aecom.yu.edu).

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.


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