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4 ATA) increases membrane conductance and firing rate in the rat solitary complex
1Department of Physiology and Biophysics, Environmental and Hyperbaric Cell Biology Facility, and 2Department of Community Health, Wright State University School of Medicine, Dayton, Ohio 45435
Submitted 20 September 2002 ; accepted in final form 4 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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4 ATA) also increase neuronal excitability. The effect of hyperbaric
helium, which mimics increased hydrostatic pressure, was determined on
putative CO2/H+-chemoreceptor neurons in the solitary
complex in rat brain stem slices by intracellular recording. Pressure
stimulated firing rate in 31% of neurons (barosensitivity) and decreased input
resistance. Barosensitivity was retained during synaptic blockade and was
unaffected by antioxidants. Barosensitivity was distributed among
CO2/H+-chemosensitive and -insensitive neurons; in
CO2/H+-chemosensitive neurons, pressure did not
significantly reduce neuronal chemosensitivity. We conclude that moderate
pressure stimulates certain solitary complex neurons by a mechanism that
possibly involves an increased cation conductance, but that does not involve
free radicals. Neuronal barosensitivity to 4 ATA may represent a
physiological adaptive response to increased pressure or a pathophysiological
response that is the early manifestation of high-pressure nervous
syndrome. brain slice; intracellular recording; cardiorespiratory control; high-pressure nervous syndrome; hyperbaric helium; hypercapnia; hyperoxia; neuron
By contrast, it is generally believed that pressures at <15 ATA have no effects on neurons in the mammalian CNS (23, 26) and that any changes noted in the CNS at these moderate pressures must be due to increased partial pressure of O2, CO2, or N2. Few studies, however, have tested whether moderate levels of pressure alter neuronal function in the mammalian CNS (13). Moreover, several recent studies suggest that small-to-moderate levels of pressure, well below 10 ATA, can alter function at the invertebrate neuromuscular junction (8, 9) and of neuronal cells in a variety of tissues (28, 39). Consequently, it becomes important to reevaluate the range of neuronal barosensitivity (i.e., sensitivity to pressure) expressed by neurons in the mammalian CNS. In our preceding paper (33), we reported that hyperbaric hyperoxia (HBO2) at 2.2-3.3 ATA increased firing rate and input resistance (Rin) of putative central CO2/H+ chemoreceptors in a cardiorespiratory control region of the brain stem known as the solitary complex (SC). This excitatory effect of hyperoxia was shown to be due to increased production of oxygen free radicals. However, it is also possible that increased pressure had an additional effect on neuronal excitability that occurred independently from that due to increased tissue PO2 and increased O2 free radicals. In the present study, therefore, we test the hypothesis that SC neurons are stimulated by moderate pressure in the absence of changes in tissue PO2, PCO2, and pH.
There were three goals in this study. First, to describe the effects of
pressure per se (2-4 ATA) on neuronal firing rate and Rin
in SC neurons. Second, to determine whether putative
CO2/H+ chemoreceptors in the SC, which are stimulated by
HBO2 (33), are
likewise affected by pressure alone and, if so, whether pressure decreases
neuronal CO2/H+ chemosensitivity.
CO2/H+-chemosensitive neurons in the SC are thought to
participate in cardiorespiratory control
(40). Previous studies have
shown that high levels of pressure (>>15 ATA) decrease respiratory drive
(44) and central
chemosensitivity (45). Third,
we wanted to determine whether barosensitivity to moderate levels of pressure,
if it occurred, was mediated by an O2 free radical-dependent
mechanism. Thom (47) reported
that hyperbaric helium (HBHe), which is often used to mimic the effects of
increased hydrostatic pressure
(33,
42-45)
and other inert gases, at pressures as low as 2.8 ATA, increases production of
O2 free radicals (i.e., superoxide). Because we have demonstrated
that CO2/H+-chemosensitive neurons in the SC were highly
sensitive to oxidative stress (and O2 free radicals)
(33), we wanted to determine
whether the use of HBHe, similarly, in our brain stem slice preparation
resulted in increased O2 free radical production. Thus we planned
to determine the effects of the antioxidant Trolox C on the barosensitive
response of SC neurons. Our findings show that hyperbaric pressures (4
ATA) increased firing rate and decreased Rin in certain SC
neurons in a reversible and repeatable manner by a mechanism that is distinct
from stimulation of the same neurons by HBO2 (i.e., O2
free radicals) and CO2 (decreased intracellular pH). These data
indicate that much lower levels of hyperbaria affect the mammalian CNS than
previously presumed. The physiological significance of neuronal
barosensitivity to moderate pressure may represent a physiological adaptive
response to increased ambient pressure or a pathophysiological response that
is the early manifestation of HPNS. Preliminary reports of these data were
already published (31,
34).
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Test Conditions
Compression medium. As in previous studies (9, 16, 17, 19, 42-45), helium was chosen as the compression medium to mimic hydrostatic pressure. All inert gases exert narcotic actions that are directly related to their lipid solubility (2, 6, 13). Helium is of very low lipid solubility (2), and, for this reason, the narcotic effects of helium are reported to be negligible, except at very high pressures, >>100 ATA (13, 36, 46), which are much higher than the levels of PB used in this study. Thus any changes in neuronal excitability, as determined by a change in firing rate during HBHe (i.e., barosensitivity), were attributed to the effects of pressure per se rather than the narcotic effects of HBHe (13).
Before compression, room air was purged from the hyperbaric chamber and
replaced with 100% helium. PO2,
PCO2, and pH of the artificial cerebral spinal fluid
(aCSF) were set at normobaric pressure (i.e., room pressure; 
1 ATA),
external to the hyperbaric chamber, and pumped into the hyperbaric chamber by
using a high-pressure liquid chromatography pump. During the ensuing helium
compression, PO2 and PCO2 in the
aCSF did not increase, because no additional O2 or CO2,
respectively, were present inside the hyperbaric chamber
(12,
13,
30,
32,
46). This is our standard test
for neuronal barosensitivity in the in vitro brain slice preparation
(13,
30,
31). Barosensitive neurons
were defined as those that responded to pressure with a 
20% change in
firing rate. In all experiments, the rates of compression and decompression
were controlled at 2 ATA/min. Brain slice temperature was maintained at
37 ± 0.5°C during helium compression and decompression
(12).
HBO2 and hypercapnic perfusates. The
details for preparation of hyperoxic and hypercapnic solutions are given in
the preceding paper (33).
Briefly, hyperbaric oxygenated perfusate (i.e., HBO2) with
normocapnia was made by equilibrating aCSF with 95 or 98% O2 at 2.2
or 3.3 ATA in separate high-pressure sample cylinders to produce
PO2 values in the aCSF of 1,640 and 2,470
Torr, respectively. PCO2 in the aCSF was kept constant
at 40 Torr by reducing the percent CO2 from 5 to 2 and 1.65%
at 2.2 and 3.3 ATA, respectively
(12,
32,
33). Normobaric and hyperbaric
hypercapnic media were made by equilibrating aCSF with 85% O2-15%
CO2 at normobaric pressure to produce media
PCO2 of 114 Torr
(10,
11,
18,
33).
Antioxidant perfusate. The antioxidant used was Trolox C (Sigma-Aldrich). Trolox C is a water-soluble analog of vitamin E, which is thought to cross the lipid bilayer, where it acts as an effective antioxidant (15) and is capable of repairing some types of oxidative damage (3). The concentrations of Trolox C used in this study ranged from 100 to 200 µM, which have been shown to block the electrophysiological effects of hydrogen peroxide on hippocampal neurons (37) and HBO2 on SC neurons (33).
Analysis and Data Presentation
Electrophysiology was done by using sharp-tipped intracellular
microelectrodes. The electrophysiological properties that were measured and
method of data collection were as described in our preceding paper
(33). Paired-sample
t-tests (P 0.05) were used to determine whether the
average firing rate, Rin, or amplitude of
afterhyperpolarizing potential (AHP) responses to pressure differed (i.e.,
mean population differences during HBHe compared with 1 ATA control)
significantly from zero. Contingency tables were used to compare tabulated
electrophysiological properties, as well as the incidence in SC neurons of
CO2/H+ chemosensitivity, HBO2 sensitivity,
and barosensitivity. The significance of associations between parameters was
determined by using 
2 or Fisher's exact test with the Yates
continuity correction, when appropriate (P 0.05). All data are
presented as means ± SE. Pressure was reported in ATA, and specific gas
tensions in the aCSF and tissue (e.g., PO2,
PCO2) are reported in Torr, where 1 ATA is equal to 760
Torr.
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RESULTS |
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(range 36-222 M). Barosensitivity
In a typical experiment, an intracellular recording was established under
control conditions at room pressure. After 10-30 min of intracellular
recording, the hyperbaric chamber was flushed and compressed to 2-4 ATA (mode
= 3 ATA) with 100% helium. In the absence of any changes in
PO2 and/or pH of the perfusate
(12,
30,
32), this level of pressure
caused, at most, a slight depolarization (3 mV), which was not always
evident, and significantly (20%) increased firing rate in 32 of 102 (31%)
neurons (Fig. 1, A and
B). The barosensitive neurons had a firing rate response
that typically occurred in conjunction with a significant decrease in
Rin [t-test, change (
) in
Rin = -7.4 ± 3.2 M; n = 23 neurons]
(Fig. 1C). In
addition, pressure decreased the amplitude of the AHP from 22.7 ± 1.1
by -1.7 ± 0.6 mV (t-test, P < 0.05, n =
22; Fig. 1D). This
type of barosensitive response was usually reversible on decompression back to
1 ATA. However, 41% of the barosensitive neurons showed an initial firing rate
that occurred with decreased Rin and decreased AHP, but
that adapted back to control levels within 5 min of reaching the maximum level
of pressure (Fig. 2). The
remaining 69% of SC neurons tested were baroinsensitive, i.e., they did not
increase firing rate in response to helium compressions up to 4 ATA
(Fig. 3, A and
B). Similarly, baroinsensitive neurons did not show a
significant change in Rin (t-test, P
> 0.25, Rin = -1.1 ± 3.5 M,
n = 44) or AHP amplitude (t-test, P > 0.25,
AHP = 0.08 ± 0.3 mV, n = 53) while at pressure
(Fig. 3, C and
D). These results indicate that PB between 2
and 4 ATA stimulates excitability in a subpopulation of SC neurons.
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Intrinsic Barosensitivity
To determine whether SC neurons are intrinsically barosensitive or if
barosensitivity is mediated by pressure-induced increases in excitatory
synaptic transmission and/or decreases in inhibitory synaptic transmission
(16,
17,
19,
22,
50), firing rate was measured
in response to increased pressure in a solution that blocked chemical synaptic
transmission (high Mg2+-low-Ca2+
medium). Previously, our laboratory has reported that this medium reversibly
blocked evoked synaptic potentials in similar brain slice preparations
(10). The direct effects of
the synaptic block medium on SC neurons include increased firing rate and
increased Rin. Changes in these parameters were used as an
indicator that high-Mg2+-low Ca2+
medium was effective in our preparation
(10).
Figure 2, A and
B, shows an example of a neuron that was transiently
stimulated by 3 and 4 ATA in control medium. We then incubated the slice for
30 min in synaptic block medium before testing for intrinsic
barosensitivity. In synaptic block medium, the cell's spontaneous firing rate
increased at normobaric pressure, presumably due to decreased
Ca2+-dependent K+ conductance and/or removal
of inhibitory input (10).
Regardless, exposure to pressure caused an additional increase in firing rate
of a similar magnitude and transient nature as that in control medium
(Fig. 2). Our results show that
the firing rate response of five barosensitive neurons in control medium
(firing rate = 1.39 ± 0.31 impulses/s) was retained in synaptic
block medium (firing rate = 3.6 ± 2.25 impulses/s), thus
indicating that these neurons were intrinsically barosensitive. These
experiments, however, did not determine the effects of pressure on synaptic
activity, which has been reported in other CNS areas at PB >35
ATA (16,
17,
50).
Barosensitivity, HBO2 Sensitivity, and CO2/H+ Chemosensitivity
We reported in the preceding paper (33) that 90% of SC neurons sensitive to HBO2 and/or chemical oxidants were also CO2/H+ chemosensitive, whereas only 19% of HBO2-insensitive neurons were CO2/H+ chemosensitive. Therefore, we wanted to determine whether pressure similarly affected preferentially HBO2- and CO2/H+-chemosensitive neurons in the SC.
Pressure and CO2/H+ chemosensitivity. To determine whether 2-4 ATA affected CO2/H+-chemosensitive neurons and CO2/H+ chemosensitivity, two types of experiments were conducted. First, we exposed cells to normobaric hypercapnic acidosis and to pressure separately to see whether there was a relationship between CO2/H+ chemosensitivity and barosensitivity. Second, to determine whether pressure affects CO2/H+ chemosensitivity (45), we compared the firing rate response of SC neurons to normobaric hypercapnic acidosis vs. hyperbaric hypercapnic acidosis.
In the first experiment, we found, as previously reported
(10), that
CO2/H+-chemosensitive neurons responded to normobaric
hypercapnia with increased firing rate and increased Rin
(Fig. 4A). We found
that, of the 58 neurons exposed to normobaric hypercapnic acidosis, 30 (52%)
were CO2/H+ chemosensitive; of these 30
CO2/H+-chemosensitive neurons, 10 neurons were also
barosensitive (Fig. 4, A and
C). The remaining 20
CO2/H+-chemosensitive neurons, however, did not respond
to pressure (Fig. 4C).
Of the 28 neurons that did not respond to CO2/H+, 9 were
barosensitive (Fig.
4B), whereas 19 did not respond to either
CO2/H+ or pressure
(Fig. 4C). These
results indicate that, whereas some
CO2/H+-chemosensitive neurons were also barosensitive,
there was not a significant association between barosensitivity and
CO2/H+ chemosensitivity (2 analysis,
P = 0.854). In other words, barosensitive neurons included both
CO2/H+-chemosensitive and
CO2/H+-chemoinsensitive neurons. Similarly, there was
not a significant association between CO2/H+
chemosensitivity and either sustained or transient barosensitivity (Fisher's
exact test).
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In the second experiment, we compared the firing rate response to hypercapnic acidosis at 1 and 3 ATA (aCSF PCO2 was set external to the hyperbaric chamber, and thus helium compression did not change pH of the aCSF; Refs. 12, 32). We found that pressure had no effect on CO2/H+ chemosensitivity (Fig. 5A), and firing rate increased by the same amount in response to CO2/H+ at 1 and 3 ATA (Fig. 5B). These results indicate that CO2/H+ chemosensitivity of SC neurons is unaffected by 2-4 ATA of hyperbaric pressure.
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Pressure and HBO2 sensitivity. In the
preceding paper, we report that increased PO2 stimulates
some SC neurons (i.e., HBO2 sensitive), specifically, putative
CO2/H+ chemoreceptors
(33). Therefore, to determine
whether pressure per se, in addition to increased PO2,
stimulates HBO2-sensitive neurons, we compared the firing rate and
Rin responses of SC neurons exposed to pressure and
HBO2. Of the 32 barosensitive neurons, 14 were HBO2
sensitive. Of the 70 baroinsensitive neurons, 30 were HBO2
sensitive. These results indicate that, whereas some barosensitive neurons
were also HBO2 sensitive, not all barosensitive neurons were
HBO2 sensitive. Thus there was not a significant association
between barosensitivity and HBO2 sensitivity (2
analysis, P = 0.896). Similarly, there was not a significant
association between HBO2 sensitivity and either sustained or
transient barosensitivity (Fisher's exact test). This was not surprising,
because the preceding analysis had revealed the lack of association between
barosensitivity and CO2/H+ chemosensitivity
(Fig. 4), and, as already
reported (33), HBO2
and CO2/H+ sensitivity are strongly associated.
It has been reported that 2.8 ATA helium, and other inert gases, can increase the production of O2 free radicals in solutions containing either xanthine oxidase-hypoxanthine or phenazine methosulfate-NADH (47). Therefore, our final goal was to determine whether O2 free radicals contribute to barosensitivity. We tested whether exposure to the antioxidant Trolox C would block barosensitivity as tested by using HBHe. Figure 6 shows that the firing rate (Fig. 6A) and Rin (Fig. 6B) responses of a barosensitive neuron were retained in the presence of Trolox C. Figure 6 also summarizes the firing rate (Fig. 6C) and Rin (Fig. 6D) responses of four neurons to HBHe in control and Trolox C media. Notice that exposure to Trolox C had no effect on the increased firing rate response to HBHe. These results indicate that the excitatory effects of HBHe on SC neurons are not mediated by O2 free radicals.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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5 min of achieving maximum pressure, whereas
the other population of barosensitive neurons maintained a sustained increase
in firing rate for the duration of the compression. In addition, we showed
that barosensitive neurons in the SC represented a heterogeneous group of
cells that included HBO2-sensitive and
CO2/H+-chemosensitive neurons
(33), which are thought to
function as central CO2/H+ chemoreceptors
(10,
11,
18,
35,
40), as well as neurons
insensitive to either increased O2 or increased
CO2/H+. HBHe as a Compression Medium
As in previous studies (33,
42-45),
helium was used to compress the hyperbaric chamber and tissue bath, thereby
effectively pressurizing the brain slice submerged in aCSF. In general, the
effects of HBHe on cells, tissues, and intact organisms are believed to be
comparable to the effects of hydrostatic pressure over a range of mechanically
tolerable pressures (6,
13). Thus we conclude that
using helium to compress the tissue bath effectively mimics hydrostatic
compression at PB 4 ATA and that any increase in neuronal
excitability observed during exposure to HBHe reflects the effects of pressure
per se and not possible narcotic actions of increased tissue helium partial
pressure (12,
30). In addition, our results
using the antioxidant Trolox C suggest that HBHe does not increase
O2 free radical production as previously proposed
(47). Dean et al.
(13) and others
(6,
23) have reviewed the evidence
supporting the use of HBHe at low levels of compression to mimic pure
hydrostatic compression.
Barosensitivity of SC Neurons
Discovering that moderate levels of pressure (2-4 ATA) stimulated
approximately one-third of the SC neurons is an exciting finding; previously,
it was thought that hyperbaric pressures <15 ATA had no effect on neurons
unless accompanied by an increase in tissue PO2,
PCO2, and/or N2 partial pressure
(23,
26,
43). Moreover, the fact that
the remaining two-thirds of SC neurons tested did not show changes in firing
rate, Rin, or AHP during exposure to 4 ATA pressure
indicates that certain SC neurons are more barosensitive than other neurons.
We anticipate that compression to pressures >4 ATA, when tested, will
stimulate firing rate and decrease Rin to a greater extent
and/or in an even greater proportion of SC neurons. This hypothesis is
supported by the observation that the effects of pressure on Na+
current increased linearly in other types of neurons with compression up to
101 ATA (42). Similarly,
previous studies showed that 30 ATA increased the number of evoked action
potentials in crayfish claw nerves
(24,
25), and pressures up to 360
ATA increased the spontaneous activity of Helix pacemaker cells, with
a corresponding 30% decrease in Rin
(48).
The membrane and synaptic mechanism(s) by which 2-4 ATA pressure increases
excitability of individual neurons remains to be determined. We did determine,
however, that neuronal barosensitivity is an intrinsic property of SC neurons
that is retained during chemical synaptic blockade in
high-Mg2+-low-Ca2+ medium. We did
not determine the effects of pressure on synaptic transmission in this study,
but we anticipate that it will also be affected by increased pressure.
Previous studies (16,
17,
50) have shown that
PB 35 ATA decreased both excitatory and inhibitory synaptic
transmission in mammalian neurons.
The physiological significance of neuronal barosensitivity to moderate levels of physical pressure is unknown. To speculate, ambient pressure, like ambient temperature, is a normally occurring environmental stimulus, and barosensitivity may represent a normal neuronal response, much like thermosensitivity (4), which contributes to how an organism responds and adapts to its changing environment. It is also possible that barosensitivity is a cellular property that occurs over a continuum of pressure, in which sensitivity to ambient pressures up to 4 ATA represents the early component of what is ultimately known as HPNS. HPNS is a complex neurological response to pressure per se that occurs in divers breathing heliox and trimix at ambient pressures >15 ATA (1, 13, 38). Symptoms of HPNS have been attributed to increased excitability of the mammalian CNS, but the exact mechanisms involved are unknown (23). Our findings are consistent with a pressure-induced increase in excitability of neurons, however, at hyperbaric pressures well below 15 ATA. It may be that, as ambient pressure increases, neuronal excitability likewise increases, along with recruitment of barosensitive neurons with higher thresholds of sensitivity (42). Once a critical threshold is reached (i.e., stimulation of critical numbers of neural networks), then symptoms of HPNS occur.
We also determined that 41% of the barosensitive responses were transient in nature; i.e., increased firing rate and decreased Rin during compression returned toward normobaric control levels within 5-10 min of sustained pressurization. Transient barosensitivity has been reported previously in Helix cells in which 50-156 ATA initially stimulated firing rate, after which the firing rate response adapted to control values within 5 min at pressure (49). Similarly, transient barosensitivity has also been observed in vivo; after 2 wk at 80 ATA, the convulsion threshold of mice increased by 35 ATA (5). The ability of many neurons to adapt to sustained pressurization may explain why previous intracellular recording studies of hippocampal neurons, which compared neurons sampled at different steady-state pressures, did not find a significant effect of pressure on neuronal excitability (43).
Cellular Mechanism of Barosensitivity
We anticipate that neuronal barosensitivity will result from the summed effect of pressure on multiple ionic currents, which, overall, are observed as a net increase in membrane conductance (i.e., decreased Rin). Hyperbaric pressure increased excitability and decreased Rin, which suggests that pressure increases an inward cation conductance (possibly Na+ or Ca2+) or possibly an outward Cl- conductance. However, other cellular signaling mechanisms may contribute to barosensitivity. A previous study found that 70 ATA evoked a depolarizing net inward current in vertebrate neurons (20); however, the observed depolarization may have resulted from a partial block of the Na+-K+-ATPase (21). Therefore, it remains to be determined which specific ion channels or transporters are affected by moderate levels of hyperbaric pressure, or even how pressure alters ion conductance and synaptic transmission in the SC. In addition, our finding that pressure reduced the AHP amplitude suggests that pressure decreases a Ca2+-dependent K+ conductance. This finding is consistent with a previous study that showed that the slow AHP measured in rat hippocampal neurons was decreased by pressures up to 100 ATA (43). In contrast, 900 ATA of hydrostatic pressure increased the open probability of large-conductance Ca2+-dependent K+ channels in chromaffin cells by 30 times (26).
It is conceivable that small hyperbaric pressures, even fractions of an atmosphere, may affect electrical signaling by neurons. At <4 ATA, it is unlikely that the changes in neuronal activity are due to changes in thermodynamically driven equilibria (13, 27). Alternatively, Macdonald and Fraser (27) have proposed that various types of cells (nonneuronal) respond to small hyperbaric pressures by a mechanical process, including cytoskeletal rearrangement and/or the development of localized shear forces, resulting from the differential compressibility of various adjoining cellular components. Moderate levels of hyperbaric pressure may similarly affect mammalian neurons. Evidence supporting this hypothesis in the mammalian CNS, however, is presently lacking (13).
Barosensitivity, CO2/H+ Chemosensitivity, and Cardiorespiratory Control
We showed that a small proportion of barosensitive neurons in a cardiorespiratory control region of the brain stem was also stimulated by hypercapnic acidosis. CO2/H+-chemosensitive neurons are thought to function as central chemoreceptors for the cardiorespiratory system (10, 11, 18, 35, 40). Although there was a trend for the CO2/H+-chemosensitive responses to decrease during hyperbaric pressure, the firing rate responses to CO2 at 1 and 3 ATA were not significantly different. However, this negative finding should be interpreted with caution, because larger levels of pressure may have a more pronounced effect on neuronal excitability. For example, Tarasiuk and Grossman (45) have shown that 100 ATA decreased central CO2/H+ chemosensitivity and the neural drive for breathing (44) in the neonatal rat brain stem-spinal cord preparation. Therefore, our finding that some SC neurons were also sensitive to moderate levels of pressure may represent the early effects of pressure on cardiorespiratory control, which precede abnormal function observed in vivo at higher pressures, e.g., high hydrostatic pressure has been shown to decrease heart rate and cause dyspnea in cats (7, 14) and reduce CO2 chemosensitivity in humans (29).
Barosensitivity, Hyperoxia, and Oxygen Free Radicals
A previous study reported that inert gases, including helium, at pressures similar to the level of hyperbaric pressure used in this study, increased production of O2 free radicals in solutions containing either xanthine oxidase-hypoxanthine or phenazine methosulfate-NADH (47). No study, however, has ever reported that inert gases at hyperbaric pressure increase O2 free radical production in isolated preparations of the mammalian CNS. Regardless, we were concerned that HBHe may increase neuronal excitability, in part, by increasing the production of O2 free radicals. This was a concern, because we reported in the preceding paper that certain SC neurons are highly sensitive to oxidative stress (33). However, the antioxidant Trolox C, which blocks sensitivity of SC neurons to HBO2 (33), did not significantly alter barosensitivity of SC neurons. Thus our results indicate that the excitatory effect of HBHe on firing rate of SC neurons was not dependent on O2 free radicals. Moreover, we showed that there was no association between HBO2 sensitivity, a highly oxidative condition (30, 31, 33), and barosensitivity in the SC. In addition, HBO2 and pressure had opposite effects on Rin; HBO2 stimulated firing rate and increased Rin (33), whereas HBHe stimulated firing rate but decreased Rin. Collectively, these findings indicate that the cellular effects of HBHe are not mediated by increased levels of O2 free radicals, as previously suggested (47), but rather are due to the effects of physical pressure (13).
In conclusion, our findings demonstrate, for the first time, that moderate
levels of pressure (2-4 ATA) increase excitability of a subpopulation of SC
neurons. The specific cellular mechanism by which increased pressure increases
neuronal excitability remains to be determined; however, it would appear that
more than one ionic current is involved. Our findings indicate that, at least
in some SC neurons, the effects of hyperbaric gases at 4 ATA, whether it
is air or a mixture of O2 and inert gas, may also include the
effects of pressure alone. Thus it will be important in future studies to
establish the effects (or lack thereof) of both physical pressure and the
chemical effects of high-gas partial pressure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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FR, impulses/s) show that
compression to 3 atmospheres absolute (ATA) with 100% helium increased FR.
B: representative membrane potential (Vm) traces
show that the recording was healthy and stable during helium compression and
decompression. C: expanded mean Vm traces
(n = 3) during hyperpolarizing current injection of -0.2 nA for 150
ms show that 3 ATA helium decreased Rin and stimulated FR.
B and C: traces 1-3 were taken at the times
indicated in A. D: average of 5-10 spontaneous action potentials
measured at 1 and 3 ATA was superimposed to show that pressure significantly
decreased the AHP in barosensitive neurons.
