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1Departments of Pediatrics, Section of Respiratory Medicine and 2Neuroscience, University of California; and 3The Rady's Children's Hospital, San Diego, California
Submitted 3 November 2006; accepted in final form 28 December 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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).
The effects of hypercapnia have long been studied (Brown 1930; Sieker and Hicham 1956), but the effects on neural properties are not very well understood. Although we have previously studied neurons from chronically elevated CO2 (8%) (Gu et al. 2004), we do not know to what extent the magnitude and duration of this elevated CO2 will have on neuronal function. We therefore asked how would neuronal properties change if we increase the duration of CO2 exposure? How would 8% CC treatment compare with a higher level of CO2, such as 12%? And what is the effect of chronic hypercapnia on neuronal function, such as excitability? In this work, we undertook this task and used 8 and 12% CO2 treatment with a duration of 4 wk to extend our previous results. We used electrophysiological and Western blot methods to determine the cellular mechanisms that were altered in response to hypercapnia. We focused our work on the hippocampus since our previous work was done on the same neurons and to compare with results from the literature (Gu et al. 2000).
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METHODS |
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A computer-controlled chamber (OxyCycler, Reming Bioinstruments, Redfield, NY) was used for the induction and maintenance of chronic CO2. The CO2 level was monitored and controlled by the software from OxyCycler at constant concentration of 8 or 12%. Animals exposed to CO2 are referred to here as CC 8% or CC 12% CO2. Oxygen in the chamber was kept at constant concentration of 21% balanced by nitrogen. Mice were placed in the chamber at the age of 2 days with their dam and were exposed to CO2 for 
25–30 days until the time of death. They hyperventilated but they did not seem to be in any distress. All exposed mice, survived the period of exposure. Control mice were born and raised in our animal facilities. Pregnant mice (CD1) were purchased from Charles River Laboratories, Wilmington, MA.
Preparation of CA1 cells
Mice at age of 26–31 days were used and killed after inhalation of isoflurane (Baxter, Deerfield, IL or VedCo). Their hippocampi were quickly removed in ice-cold dissection solution and sliced into transverse sections with thickness of 400 µm. The slices were immediately transferred to a home-made container with 10 ml of fresh, oxygenated, and slightly stirred HEPES buffer at room temperature. After 30 min of exposure to protease (0.1%, Sigma) digestion, and 20 min of trypsin (0.08%, type XI, Sigma), 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 volume (0.25 ml) of HEPES buffer with three pipettes of gradually reduced openings. The Albert Einstein College of Medicine Animal Care and Use Committee and University of California San Diego IACUC (Institutional Animal Care and Use Committee) have 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, Sarasoda, FL). The electrodes were fire-polished, and resistances were 2–5 M
for voltage-clamp experiments and 7–9 M for current-clamp experiments measured in the solutions in the following text. Membrane potentials (Vm) and action potentials (APs) were recorded in the current-clamp mode. Input resistance (Rm) was calculated at –70 mV as 1/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-square regression analysis for 100 data points between voltage and current. Current traces in voltage clamp were leak-subtracted. Liquid junction potentials were nulled for each individual cell with the Axopatch 1C amplifier.
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 and pH was 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 and pH was adjusted to 7.4 with Tris. The external solutions for the voltage-clamp experiments contained similar reagents as in the current-clamp experiments except for 10 mM TEA chloride, 5 mM 4-AP, and 0.1 mM CdCl2 and reducing NaCl from 130 mM to 117 mM. The internal pipette solution for the voltage-clamp experiments was also similar to the internal solution for the current-clamp experiments except for the use of either CsF instead of KCl [we had previously shown that there was no difference in whole cell Na+ current recorded when CsF or CsCl was used (O'Reilly et al. 1997) excitatory postsynaptic potential. Osmolarity of all solutions was adjusted to 290 mosM. All recordings were performed at room temperature (22–24°C). One-tailed Student t-test was performed for comparisons. Significances were indicated if P < 0.05 assuming two groups had an equal variance. Numbers in the text and in the figures were given as means ± SE. All chemicals were purchased from Sigma.
Recording criteria
These criteria have been previously used, as detailed in our previous publications (O'Reilly et al. 1997). Briefly, we have used morphologic and electrophysiological criteria. Morphologic criteria: CA1 cells were used if they had a smooth surface, a three-dimensional contour, and pyramidal shape. Similar criteria have been used by us (Gu and Haddad 2003; Gu et al. 2000) and others (Hamill et al. 1981) on freshly dissociated neurons. The CA1 neurons studied were obtained from 12- to 17- or 26- to 31-day-old mice. Electrophysiological criteria: 1) neurons were considered for recording if the seal resistance was >1 G. 2) Only neurons with a holding current of <0.1 nA (command potential –100 mV) were used in the study. 3) Series resistance was <10 M in neurons studied. The series resistances were compensated at 90% level with the Axopatch 1C amplifier (Axon Instruments). Under these conditions, the error caused by uncompensated series resistances was <0.6 mV. The error was not corrected. To obtain adequate voltage clamp and minimize the space-clamp problem, only neurons of small size with short processes were used in the current measurement. In addition, only cells with current-voltage curves that were smoothly graded over the voltage range of activation (approximately –50 to –10 mV) were used, as we have done in the past (Gu and Haddad 2003; Gu et al. 2000).
Immunoblotting
Hippocampi from four different animals from each of the three groups (CON, 8% CC, and 12% CC; each group had four samples) to lysis buffer (200 mM mannitol, 80 mM HEPES, 41 mM KOH, 1 tablet/50 ml of Complete protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany), 230 µM phenylmethylsulfonyl fluoride, pH 7.5). Samples were homogenized and the homogenate was centrifuged at 1,000 g at 4°C for 10 min. The supernatant was then centrifuged at 100,000 g at 4°C for 1.5 h. The pellet was re-suspended in lysis buffer, and protein concentration was determined with the use of the Bio-Rad Dc Protein Assay kit. Membrane proteins (30 µg) were resolved on 4–12% precast NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) and electro-transferred 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, Saint Louis, MO) were applied. Protein signals were detected with the use of an enhanced chemiluminescence system (ECL; Amersham, Chalfont, UK). For normalization, membranes were stripped and re-probed with affinity-purified goat polyclonal antibody to actin at 1:500 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.
Statistical analysis
Student's t-test or the Wilcoxon rank sum test was used for comparisons. All values represent means ± SE. Differences in means are considered significant if P < 0.05.
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RESULTS |
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To determine whether CO2 affects neuronal excitability, we measured the rheobase. Stepping from the same Vm (–75 mV) in the current-clamp mode, the current required to generate one AP was 64 ± 11 pA (n = 9) at 8% and 73 ± 8 pA (n = 9) at 12%, values that were 36 and 55% higher than those of CON (47 ± 12 pA, n = 8), respectively (Fig. 1, A–C), with P values of 0.27 and 0.046 when CON was compared with 8 and 12% (Fig. 1D). Samples of voltage traces from CON and 8 and 12% CC are shown in Fig. 1.
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Because CC treatment decreased neuronal excitability in 12%-treated neurons, we then investigated the potential factors that caused the lowering of excitability. We began with the passive neuronal properties such as resting membrane potential, Vm and input membrane resistance, Rm. CC-treated animals (8 and 12%) for 28 days did not show any effect on Vm. Indeed, CA1 neurons had similar membrane potentials as those of CON neurons (Vm: –37 ± 4 mV, n = 10, and –36 ± 3 mV, n = 17 for CC at 8 and 12% CO2 vs. –42 ± 2 mV, n = 10, for CON). Furthermore, CC treatment did not have any effect on Rm and whole cell capacitance.
Na+ current magnitude
Because 12% CO2 treatment significantly changed neuronal excitability but did not change passive neuronal properties of CA1 hippocampal neurons, we then asked whether the Na+ channel properties had been affected by the treatment. We first examined the effect of CC treatment on the Na+ current of both CON and CC 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 zero current. TTX (1 µM) blocked that current. After 28 days of CC treatment, the average peak Na+ current was 1,971 ± 662 pA (n = 17) for 8% as compared with control (1,465 ± 209 pA, n = 22, P = 0.21). CC treatment at 12% level, however, dramatically suppressed the peak Na+ current (882 ± 129 pA, n = 17) as compared with the Na+ current in CON (P = 0.02). To eliminate the role of cell membrane surface area, we divided the peak current by cell capacitance. After 4 wk of chronic CO2 exposure, the current density at 8% (424 ± 107 pA/pF) was similar to that of control (436 ± 56 pA/pF, P = 0.56). However, the current density at 12% (244 ± 36 pA/pF) was substantially reduced when compared with the control group (P = 0.005) (Fig. 2E).
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Because CC at 12% treatment decreased the Na+ current density, we then asked whether other Na+ current gating properties have been affected. We examined the gating properties of the Na+ current to determine whether other properties could have played a role in determining the difference in excitability.
ACTIVATION. Holding neurons at –130 mV and stepping commands from –70 to 10 mV in 10-mV increments, we obtained a family of curves of Na+ currents at different Vm. Their I-V curves are illustrated in Fig. 3. When the relative conductance-voltage curves were plotted and fitted to the Boltzmann equation, we found that after 4 wk of CC treatment, the curve was shifted slightly in a depolarization direction at 8 and 12% as compared with that in the control group. Neither the mid-point of the curves nor the slope factors for the CO2 treatment were different from these of control (Fig. 3).
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RECOVERY FROM INACTIVATION.
When we examined the recovery from inactivation, the currents evoked by the second pulse were smaller than the those of first one with their amplitude increasing with the increased intervals between the two pulses (Fig. 4). The time constants (
h) for recovery from inactivation could be fitted by a first-order exponential equation. When h from both 8 and 12% neurons were compared with control, these were not different from those for CON (5.7 ± 0.9 ms, n = 11, 7.2 ± 2.0 ms, n = 5, and 5.8 ± 0.5 ms, n = 14, for CC at 8%, CC at 12%, and CON neurons, respectively; Fig. 4D). However, when we compared individual data points with each other, our data (<20 ms) showed significant differences between 12% CO2 and control (Wilcoxon rank sum test).
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d at –100 mV, could be fitted by a first-order exponential equation. After 4 wk of treatment, d was smaller, albeit not significantly, in both CC 8 and 12% neurons as compared with the CON. NA+ CHANNEL EXPRESSION. We further examined whether the decrease in Na+ current density is due to a decrease in Na+ channel expression and, if so, which Na+ channel subtype might be involved. Because there are three major neuronal subtypes of Na+ channels in the CNS, we used specific antibodies against each neuronal isoform of Na+ channel and performed immunoblotting assays. Mice exposed to 8% CO2 for 4 wk did not show any change in the hippocampal membrane protein expression of any of Na+ channel subtypes type I–III Na+ channel subtypes. However, exposure to 12% CO2 selectively decreased Na+ channel subtype I and III protein level by 27 and 20% respectively (n = 4; P = 0.026 and P = 0.029; Fig. 5). It is interesting to note that the downregulation of Na+ channel expression was less than the decrease in Na+ channel current density, i.e., reduced to 44% of control, in contrast to approximately one-half of this decrease in the expression of membrane Na+ channel.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Because the Na+ channel gating properties, with the exception of the slow-down of the recovery from inactivation, did not change by CO2 treatment, the decreased excitability at 12% is most likely due to the lower Na+ current density CO2 treatment induced. One hypothesis for this decrease in Na+ channel density is that the Na+ channel protein expression is decreased. This is borne out by our findings of decreased expression of Na+ channel subtypes (I and III).
It is important to highlight that the difference in excitability is most likely not related to the major Na+ channel gating properties because these have not been altered by the CO2 treatment. The slowing down of recovery from inactivation could be only a partial explanation as firing is diminished. However, we do not believe that this can explain the difference in excitability without considering Na+ channel expression. In addition, if the Na+ current density is a product of Na+ channel expression, channel open probability (Po), single-channel amplitude and conductance, then it is still possible that Po, or single-channel amplitude might change and could play a role. By itself, the change in Na+ channel expression can account for a major part of the change in cellular excitability, but this does not rule out other factors.
Another important conclusion from this work is that different results have been obtained from different CO2 treatments. Indeed, this is not surprising as different concentrations of CO2 have been reported to have totally opposite effects on Kir4.1 and Kir5.1 channels. For example, 3% CO2 enhanced Kir currents and caused hyperpolarization and 8% CO2 suppressed Kir currents and caused depolarization (Cui et al. 2001). Opposite effects on field excitatory postsynaptic potentials (EPSPs) were also reported with different concentrations of CO2 in hippocampal slice preparations: 2% CO2 is an enhancer, whereas 10 and 20% were inhibitors of field EPSPs (Dulla et al. 2005). This idea does not seem to be unique for the brain as different concentrations of CO2 have different effects, for example, on lung development. We have recently shown that 8% CO2 has a major effect on gene regulation, such as for surfactant proteins A and D, but 12% CO2 does not (Li et al. 2006). Studies dissecting the effect of various CO2 levels on various organs would be necessary in the future.
How this decreased excitability actually occurs after a 4-wk treatment of 12% CO2 is not clearly understood. A number of factors could have contributed. First, although acute hypercapnia is not well compensated (Bracket et al. 1965), chronic hypercapnia is generally compensated. Therefore we believe that the effects of chronic hypercapnia on hippocampal neurons are most likely due to either CO2 or HCO3–. Second, it is possible that the effect of CO2 is either direct, such as on neuronal membranes (Balestrino and Somjen 1988), or via neuromediators or modulators. Third, if the effect is indirect, CO2 effect could be induced by alterations in adenosine and ATP (Dulla et al. 2005), or alterations in growth factors (e.g., platelet activating factor) (Simakajornboon et al. 1998). Effects of CO2 via pH are controversial because it has been reported that short-term treatment with CO2/HCO3– has its effect via HCO3– and not from CO2 or pH (Bruehl and Witte 2003) and the effect of CO2 on Kir1.1 channels was secondary to changes in pHi and pHo (Zhu et al. 2000). Fourth, although we used HEPES as buffer in our experiment (pH 7.4), this might not have been the pH that exposed mice had. It is possible that their pH sensitivity might have been altered. Using CO2/HCO3– as buffer will affect neuronal excitability and Na+ channel gating properties as we have previously shown (Gu et al. 2000).
We have demonstrated in this work that chronic 12% CO2 treatment decreased the current density by 44%, and decreased Na+ channel types I and III expression by 27 and 20%, respectively. The discrepancy between current density and protein expression might be due to several factors. For example, first, Na+ channel expression using Western blot analysis was underestimated. Na+ channel type II expression was also decreased by the treatment (as a trend with no statistical significance) and type IV, known to be in the hippocampus (Shaller et al. 1995), was not tested in our experiments. Second, the current density was based on the peak currents of whole cell recording. This peak current is not equivalent to Na+ channel expression by Western blot analysis because it is a product of the number of the Na+ channels involved, the single-channel amplitude, the single-channel conductance, and the open probability of the Na+ channels. Therefore it is possible that the open probability of the channel, for example, contributes to the peak current.
Our previous data (Gu et al. 2004) and our current results lead us to conclude that CO2 stimulates the expression of Na+ channels when CO2 exposure is early in life. By so doing, CO2 increases excitability of neurons (Gu et al. 2004; Xia and Haddad 1994). In our previous paper (Gu et al. 2004), we found that the 8% CO2 treatment over a relatively short period of time (2 wk) increased neuronal excitability over that of age-matched control neurons. In this current study, we found that a longer period of the same treatment (4 wk) did not further increase this excitability.
In summary, we have shown that 12% but not 8% CO2 decreases neuronal excitability when mice are exposed for 4 wk to this stress. This decreased excitability is secondary, by and large, to a decrease in Na+ channel subtype I and II expression.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. G. Haddad, Dept. of Pediatrics, University of California San Diego, 9500 Gilman Dr., San Diego, CA 92093-0735 (E-mail: ghaddad{at}ucsd.edu)
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REFERENCES |
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