Department of Molecular and Integrative Physiology, Neuroscience Program, and College of Medicine, University of Illinois, Urbana, Illinois 61801
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Horn, Eric M. and Tony G. Waldrop. Hypoxic Augmentation of Fast-Inactivating and Persistent Sodium Currents in Rat Caudal Hypothalamic Neurons. J. Neurophysiol. 84: 2572-2581, 2000. Previous work from this laboratory has indicated that TTX-sensitive sodium channels are involved in the hypoxia-induced inward current response of caudal hypothalamic neurons. Since this inward current underlies the depolarization and increased firing frequency observed in these cells during hypoxia, the present study utilized more detailed biophysical methods to specifically determine which sodium currents are responsible for this hypoxic activation. Caudal hypothalamic neurons from ~3-wk-old Sprague-Dawley rats were acutely dissociated and patch-clamped in the voltage-clamp mode to obtain recordings from fast-inactivating and persistent (noninactivating) whole cell sodium currents. Using computer-generated activation and inactivation voltage protocols, rapidly inactivating sodium currents were analyzed during normal conditions and during a brief (3-6 min) period of severe hypoxia. In addition, voltage-ramp and extended-voltage-activation protocols were used to analyze persistent sodium currents during normal conditions and during hypoxia. A polarographic oxygen electrode determined that the level of oxygen in this preparation quickly dropped to 10 Torr within 2 min of initiation of hypoxia and stabilized at <0.5 Torr within 4 min. During hypoxia, the peak fast-inactivating sodium current was significantly increased throughout the entire activation range, and both the activation and inactivation values (V1/2) were negatively shifted. Furthermore both the voltage-ramp and extended-activation protocols demonstrated a significant increase in the persistent sodium current during hypoxia when compared with normoxia. These results demonstrate that both rapidly inactivating and persistent sodium currents are significantly enhanced by a brief hypoxic stimulus. Furthermore the hypoxic-induced increase in these currents most likely is the primary mechanism for the depolarization and increased firing frequency observed in caudal hypothalamic neurons during hypoxia. Since these neurons are important in modulating cardiorespiratory activity, the oxygen responsiveness of these sodium currents may play a significant role in the centrally mediated cardiorespiratory response to hypoxia.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Caudal hypothalamic neurons
recorded in vivo from several different species including the rat, cat,
and rabbit are activated during brief periods of hypoxia (Cross
and Silver 1963; Dillon and Waldrop 1993
;
Ryan and Waldrop 1995
). Since this region is well known
to be involved in the modulation of cardiorespiratory function, we have
hypothesized that these neurons contribute to the increased ventilation
present with a decreased level of environmental oxygen (Horn and
Waldrop 1997
; Waldrop and Porter 1995
). Evidence from this laboratory has supported the hypothesis by demonstrating that
these neurons are inherently depolarized and show an increased firing
frequency during briefs periods of hypoxia in vitro (Dillon and
Waldrop 1992
). Furthermore this membrane response appears to be
due to a sustained inward current caused by an increased membrane
conductance through tetrodotoxin-sensitive sodium channels (Horn
et al. 1999
).
Hypoxia has recently been shown to modulate several types of ion
channels, including potassium, calcium, and sodium channels, at the
whole cell level in vertebrate central neurons (Mironov and
Richter 1998; Mironov et al. 1998
;
O'Reilly et al. 1997
). In addition, single-channel
analyses of these channels in neurons and other cell types have
demonstrated that their properties are altered during periods of
hypoxia (Fearon et al. 1997
; Jiang and Haddad
1994
; Ju et al. 1996
). Of particular interest is
the effect that hypoxia has on voltage-sensitive sodium channels since
these channels are the primary effectors of neuronal excitability. The two main currents that flow through these channels are the
fast-inactivating and persistent sodium currents (Brown et al.
1994
; Chandler and Meves 1970
; Keynes
1994
). Both of these sodium currents are modulated by hypoxia
in several cell types. In the human neocortex, fast-inactivating sodium
currents decrease significantly when subjected to a hypoxic stimulus in
vitro (Cummins et al. 1993
). Furthermore
fast-inactivating sodium currents in rat hippocampal neurons show a
similar decrease in amplitude during hypoxia that is caused by an
activation of protein kinase C (O'Reilly et al. 1997
).
Finally, single-channel analyses of sodium channels in rat ventricular
myocytes has demonstrated that the persistent sodium current is
enhanced during brief periods of hypoxia, while whole cell studies in
rat neurons have shown both increases and decreases in this current
during hypoxia (Hammarstrom and Gauge 1998
; Ju et
al. 1996
; Kawai et al. 1999
).
Since a functional enhancement of sodium currents is one possible cause of the depolarization and increased firing frequency seen during hypoxia in caudal hypothalamic neurons, the purpose of this study was to determine the effects of hypoxia on the specific properties of the two main sodium currents (fast-inactivating and persistent) in these neurons. To accomplish this goal, we voltage-clamped acutely dissociated caudal hypothalamic neurons in the whole cell patch-clamp configuration and recorded sodium currents during normoxic and hypoxic conditions. The results from these studies demonstrate an augmentation of both the fast-inactivating and persistent sodium currents during hypoxia in rat caudal hypothalamic neurons.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue preparation
All animal protocols and procedures described were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guidelines of the Laboratory Animal Care Advisory Committee at the University of Illinois. Unless otherwise noted, all pharmacological agents were obtained from Sigma Chemical, St. Louis, MO.
Caudal hypothalamic neurons from 18- to 28-day-old male Sprague-Dawley
rats (Harlan) were acutely dissociated using a modified procedure as
previously described in hypothalamic and other rat neurons
(Cummins et al. 1991; Kay and Wong 1986
;
Rhee et al. 1996
; Uteshev et al. 1996
).
Briefly, the rats were anesthetized with vaporized halothane and
rapidly decapitated. The brain from each animal was quickly removed,
and the hypothalamus was blocked according to visual cues and placed in
100% oxygenated, iced artificial cerebrospinal fluid [ACSF (in mM)
containing 120 NaCl, 1.25 NaH2PO4, 3 KCl, 2 MgCl2, 2 CaCl2, 25 Na+-HEPES, and 10 glucose at pH 7.2 and 295 mOsm,
4°C]. The hypothalamus was then cut into 500-µm-thick slices using
a tissue chopper, and the slices were transferred into an incubation
chamber containing ACSF at room temperature. The slices were incubated
in ACSF containing 0.037% trypsin (Sigma, type XI) at room temperature
for 20 min followed by three serial washes in ACSF. This was followed
by another 20-min incubation in ACSF containing 0.025% protease
(Sigma) at room temperature. The slices were then washed three times in ACSF and stored in the incubation chamber for no more than 6 h prior to recording.
Immediately prior to recording, an individual hypothalamic slice was
removed from the incubation chamber and placed in a petri dish
containing ACSF gassed with room air. Using a dissecting microscope,
the caudal hypothalamus was carefully dissected from the rest of the
hypothalamus using visual landmarks according to a stereotaxic atlas
(Paxinos and Watson 1986). The tissue was then placed
into a trituration solution (identical solution as ACSF except for the
removal of CaCl2 and the addition of 10 mM EGTA
and 2 mM kynurenic acid) and slowly triturated using a series of three
diminishing bore Pasteur pipettes with fire-polished tips. The cell
suspension was then plated onto a coated coverslip (Cell-Tak,
Collaborative Biomedical Products) and placed into the recording
chamber (RC-25, Warner Instruments) for 5 min prior to recording to
facilitate adhesion to the recording surface.
Electrophysiological recordings
Individual neurons were visualized using an inverted microscope
equipped with phase contrast optics (PIM, World Precision Instruments).
According to previously described visual criteria, only those neurons
deemed viable were recorded (Kay and Wong 1986; O'Reilly et al. 1997
). Briefly, viable neurons were
identified by the presence of a smooth membrane surface, nonprominent
nuclear complex, three-dimensional shape, at least two projections
(axonal and dendritic processes), and minimal swelling.
Whole cell recordings were made using single-electrode patch-clamping
techniques (Hamill et al. 1981). Microelectrodes
suitable for patch-clamping were pulled from borosilicate glass
(PG52165-4, World Precision Instruments) using a gravity micropipette
puller (PP-83, Narishige), and the tips were fire-polished to a tip
resistance of 1-4 M
. The intracellular solution was specifically
designed to isolate sodium currents and contained the following (in
mM): 130 CsF, 10 TEA-Cl, 11 EGTA, 1 CaCl2, 2 MgCl2, 1 ATP, and 10 HEPES, pH 7.25, 275 mOsm. In
a few neurons, the CsF was replaced with KF and the TEA-Cl removed to
record normal membrane properties. Cells were approached using a
motorized micromanipulator (DC-3K, Fine Science Tools) until a seal
resistance of
2 G
was obtained. All recordings were acquired in
the voltage-clamp mode using the AxoPatch 200A amplifier (Axon
Instruments) with the currents filtered with a 4-pole Bessel filter
(
3 dB and 5 kHz), digitized at 15 kHz, and stored on a computer for
later analysis (PClamp 6.0.5 for acquisition and both 6.0.5 and 8.0.1 for analysis). In addition, the junction potential was nullified
immediately prior to seal formation using the internal circuitry of the
amplifier. When recording fast-inactivating sodium currents, the
extracellular solution consisted of the following (in mM): 20 NaCl, 120 N-methyl-D-glutamic acid (NMDG), 3 KCl, 2 CaCl2, 2 MgCl2, 0.5 NiCl2, 0.1 CdCl2, 2 4-aminopyridine (4-AP), 10 TEA-Cl, 10 glucose, and 25 HEPES at pH 7.4 and 295 mOsm. When recording persistent sodium currents, the solution
was the same as the ACSF except for the addition of
NiCl2, CdCl2, TEA-Cl, and
4-AP in the same concentrations as the fast-inactivating sodium current
solution. Additionally, several neurons were recorded using the
standard ACSF to determine normal membrane properties. All solutions
were perfused into the recording chamber at 1 ml/min.
Once a neuron was obtained in the voltage-clamp mode, the series
resistance (80%) and capacitance were compensated using the internal
circuitry of the amplifier. Taking into consideration the large and
rapid currents that flow through the sodium channel, several
electrophysiological criteria were instituted to obtain acceptable and
reliable recordings of these currents. First, no cells were used for
data analysis if the current needed to hold the membrane at
100 mV
exceeded 100 pA. We have found that cells needing a large current to
maintain a negative holding potential have significant leaks and are
unstable. Second, only cells that were obtained with a series
resistance of <10 M
were studied because the voltage offset can be
significant (>10 mV with an uncompensated series resistance of >10
M
when recording currents ~1 nA). Last, the initial activation
curves (see following text) for voltage steps from a holding potential
of
90 mV were compared with a holding potential of
60 mV to
elucidate any voltage errors. If the activation curves differed by >5
mV, then the data for that cell was discarded.
Experimental protocol
Once a cell had been successfully patch-clamped, a series of
voltage-step protocols were performed during normoxic conditions (chamber gassed with room air), depending on the type of current to be
studied. For fast-inactivating currents, the activation and
inactivation properties of each cell were analyzed. From a holding
potential of 90 mV, depolarizing pulses of 60 ms were induced
starting at
60 mV and continuing to +25 mV in 5-mV increments. The
peak inward current at each depolarizing step was recorded, and the
voltage dependence of activation was calculated using the conductance
G at each step where G = Ipeak/Vstep
Vreversal. The reversal potential
(Vreversal) was determined by taking a linear regression of the last four data points from the current/voltage plot from each protocol. The inactivation properties were determined by
measuring the peak inward current generated by a 20-ms step depolarization to
10 mV from each of 200 ms prepulses from
100 to
+10 mV in 10-mV increments from an initial holding potential of
90
mV. When analyzing the persistent component of the sodium current, two
approaches were taken. First, long (60 ms) step depolarizations were
instituted over the same range as with the fast-inactivating current,
and the last 10 ms of current was quantified at each step to generate
the current-voltage relationship. Second, depolarization ramps from
90 to +35 mV of various rates (0.5-5.0 mV/ms) were induced and the
peak, persistent current measured for each rate. Each set of protocols
was repeated for every cell at intervals of 2-3 min to elucidate any
shift from baseline prior to testing during hypoxia. After the baseline
currents were established, the chamber was perfused with a hypoxic
solution that was identical to the control solution except that the
hypoxic solution had been rigorously gassed with 100%
N2 for
1 h and the oxygen scavenger Na2S2O4
(2 mM) was included. No shift in pH occurred with perfusion of the
hypoxic solution compared with control perfusion. In some experiments,
a Clark-style oxygen-sensing electrode (Model 737, Diamond General) and
polarographic amplifier (Model 1900, AM Systems) were used to measure
the absolute oxygen level in the chamber. The polarographic electrode
was calibrated using four separate chambers of varied known oxygen
partial pressure and generating a standard curve. At least 2 min was
allowed for the hypoxic solution to saturate the chamber before the
voltage protocols were repeated.
Data analysis
To eliminate any effects of a shift in activation between
normoxic and hypoxic conditions, the peak fast-inactivating current was
determined as the largest current over the entire range of voltage
steps (usually between 20 and +10 mV) and not just at a single step
level. This peak current was then compared between control and hypoxic
conditions using a paired Student's t-test. A Boltzman fit
of the data was used to obtain the activation curves for the
fast-inactivating current as follows:
G/Gpeak = 1/{1 + exponential[(V1/2
V)/k]},
where Gpeak is the peak conductance
value, G is the conductance for each voltage step (see
preceding text), V is the ending voltage level at each step,
V1/2 is the half-activation value, and
k is the slope constant. For the steady-state inactivation
curves, the Boltzman fit was used as follows:
I/Ipeak = 1/{1 + exponential[(V1/2
V)/k]}
where Ipeak is the peak current,
I is the current generated from the step depolarization to
10 mV from each prepulse step, V is each individual
prepulse (see preceding text), V1/2 is
the half-activation value, and k is the slope constant. For
both the activation and inactivation analyses, the
V1/2 values were compared between
control and hypoxic conditions using Student's paired t-test. The activation kinetics were determined by measuring
the time between the onset of the voltage pulse and the peak current response for each step. Inactivation kinetics were determined by
fitting the current tracings at each step using a double exponential of
the Chebyshev algorithm in Clampfit 8.0.1 (Axon Instruments).
Persistent current for the long step depolarizations was measured as the average current over the final 10 ms of the step at each voltage level. Again to eliminate any effect from a shift in activation, the peak current over the entire range for each protocol was used as comparison between normoxic and hypoxic states. The persistent current was measured during the ramp protocols as the single peak current at each ramp rate after excluding any possible fast-inactivating current. The persistent current at each rate was then compared between control and hypoxic conditions with the common rate of 1 mV/ms used to compare the sample populations due to the consistent nature of this rate to generate only persistent and not fast-inactivating currents. Data are presented as means ± SE, and the significance level for each comparison was determined to be P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recordings were made from a total of 61 caudal hypothalamic neurons with separate populations being used for analysis of the fast-inactivating (n = 28) and persistent (n = 26) sodium currents. All of the neurons studied had at least two projections extending from their soma, and most were of an either bipolar or multipolar morphology. The typical appearance of a multipolar caudal hypothalamic neuron just prior to seal formation is shown in Fig. 1A.
|
Basal membrane voltage properties (resting membrane potential = 49.0 ± 3.8 mV, input resistance = 425.0 ± 98.0 M
,
action potential amplitude = 100.2 ± 6.7 mV, and action
potential half-width = 0.96 ± 0.1 ms) were determined using
protocols in the current-clamp mode from seven separate neurons
recorded in normal ACSF. The membrane properties of these cells were
similar to previous studies of caudal hypothalamic neurons in a brain
slice preparation (Horn et al. 1999
), indicating that no
significant changes occurred due to the dissociation. One example of
the membrane responses of a caudal hypothalamic neuron to depolarizing
and hyperpolarizing pulses is shown in Fig. 1B. This cell
demonstrated an increased conductance during hypoxia as seen in the
I-V plot. Basal properties were not determined from the
other 54 neurons since the extracellular and pipette solutions needed
to isolate the separate sodium currents distorted these measurements.
The oxygen level in the recording chamber, measured with the
polaragraphic electrode, rapidly decreased to 10 Torr within 2 min of
switching the medium and reached a steady state 5 Torr within 4 min
from a baseline of ~160 Torr (Fig. 2).
This level of hypoxia has previously been shown to elicit a rapid
depolarization and increase in firing frequency in caudal hypothalamic
neurons in a brain slice preparation (Horn et al. 1999
).
|
Fast-inactivating sodium currents
The averaged results for maximal fast-inactivating sodium current
elicited by a depolarizing pulse from a holding potential of 90 mV
was 956.9 ± 149.2 pA in the 11 neurons tested with the sodium
level reduced by >85%. This current was significantly enhanced (1258.2 ± 242.8 pA) during the period of hypoxia as shown in Fig. 3. When TTX (2 µM) was introduced into
the bathing medium, the entire fast-inactivating current was abolished
(data not shown), suggesting that the currents analyzed were
exclusively TTX-sensitive sodium currents. Normoxic conditions were
restored in 6 of 11 of the cells following the hypoxic period to
determine if the changes noted were due to a shift in the baseline,
which can arise from intracellular washout in the patch-clamp
configuration. In these cells, the initial normoxic maximal
fast-inactivating sodium current of 1,138.2 ± 191.9 pA was
significantly enhanced during hypoxia (1,498.8 ± 348.5 pA) and
subsequently returned toward the normoxic level (1,339.7 ± 287.4 pA) when tested in the control conditions following hypoxia. An example
of the recovery of the fast-inactivating current following the hypoxic
perfusion is shown in Fig. 3.
|
The peak fast-inactivating sodium current was accentuated during
hypoxia throughout the entire activation range as shown in Fig.
4. This figure illustrates the rationale
for using the peak sodium current at the voltage step where the
greatest current was elicited (mainly between 10 and 0 mV). Because
the activation currents shifted during the hypoxic period, using a
fixed voltage step value (such as
90 to
10 mV) would have failed to
accurately compare the fast-inactivating current between the normoxic
and hypoxic conditions. The activations of these currents were analyzed in more detail by calculating the conductance ratio at each step and
fitting the data to the Boltzman equation. As shown in Fig. 5, the
V1/2 value was significantly shifted
to a more hyperpolarized level during the hypoxic stimulation
(
17.9 ± 1.6 vs.
21.8 ± 2.0 mV, P < 0.05), indicating a lowering of the activation threshold and a greater
ability to generate an action potential at hyperpolarized membrane
potentials (Werkman et al. 1997
). The steady-state
inactivation was also analyzed using the Boltzman fit to the current
ratio at each step throughout the entire inactivation range. Figure 6 shows that the inactivation curve
during hypoxia was significantly shifted to a more negative value when
compared with the normoxic conditions
(V1/2 =
51.4 ± 1.6 vs.
54.4 ± 2.0 mV, P < 0.05), indicating that
hypoxia leads to a greater inactivation at hyperpolarized membrane
voltages.
|
|
|
The kinetics of activation and inactivation were analyzed to determine any effect of hypoxia on these properties. As shown in Fig. 7A, the kinetics of inactivation were best fit using a double exponential that revealed a fast and slow component. When combined, the inactivation kinetics were not affected by hypoxia in these cells (Fig. 7B) nor were either the fast or slow components affected by hypoxia throughout the entire inactivation range (Fig. 7C). Likewise the activation kinetics of these neurons were unaffected by hypoxia throughout the entire range of activation as shown in Fig. 7D.
|
Persistent sodium current
The persistent sodium current in the caudal hypothalamic neurons
was differentiated from the fast-inactivating current by two methods.
First the cells were slowly depolarized from 90 to +35 mV using a
ramp protocol. Figure 8A
demonstrates the distinction between the fast-inactivating and
persistent currents using this method. The faster ramp rate (2.0 mV/ms)
elicited a fast-inactivating current with the persistent current
remaining after the fast-inactivating current ceases following
inactivation. When the ramp depolarization rate was slowed to 0.67 mV/ms, the inactivation of the fast-inactivating sodium current is
elicited at the same time as the activation of this current thus only
the persistent sodium current was observed. The average persistent
current elicited (at a ramp rate of 1 mV/ms) in 11 caudal hypothalamic
neurons perfused with the low sodium solution was 114.7 ± 23.0 pA. This current was significantly increased to 162.5 ± 27.6 during the hypoxic stimulus (Fig. 8C). When the control
solution was perfused into the chamber following the hypoxic period,
the persistent current returned to near normal levels (Fig.
8B). Inclusion of TTX (2 µM) in the chamber (Fig.
8B) abolished most of the persistent current; however, a
small (<50 pA) TTX-insensitive component remained that was minimally
apparent above the level of the noise in the recordings.
|
The second method of analyzing the persistent current is demonstrated
in Fig. 9. Long (60 ms) depolarizing
steps from a holding potential of 90 mV were induced in a solution
containing a normal sodium concentration and the current recorded
during the last 10 ms of the step. Figure 9A shows the
latter portion of the current response to the step depolarization that
corresponds to the persistent sodium current; the initial
fast-inactivating current in the initial part of the step has ceased by
this point and has been omitted in the figure. The persistent sodium
current was significantly increased from the control conditions, which
returned to near control levels when normoxia was restored following
hypoxia (Fig. 9A). Furthermore the inclusion of TTX (2 µM)
into the bath completely attenuated the persistent current in these
neurons as shown in Fig. 9A. The activation range of this
persistent current was slightly more hyperpolarized than the activation
range of the fast-inactivating current and hypoxia significantly
enhanced the current throughout the entire activation range (Fig.
9B). The magnitude of this current was relatively small
compared with the random noise, therefore activation curves were not
obtained due to the low correlation coefficients with the Boltzman
equation. When the peak persistent currents from all 10 neurons tested
were compared, there was a significant enhancement in current at the
end of the hypoxic period as compared with the end of the normoxic
period (
93.3 ± 15.5 vs.
130.6 ± 25.5 pA,
P < 0.05; Fig. 9C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study demonstrates that both the
fast-inactivating and persistent sodium currents in rat caudal
hypothalamic neurons are increased during a brief period of
hypoxia. These findings significantly enhance our understanding of
the mechanism of neuronal stimulation that occurs during hypoxia in
these cells. The observed augmentation of the sodium currents during
hypoxia may affect the neurons in this region in two ways. First, the
increased amplitude and hyperpolarized activation curve values of the
fast-inactivating sodium current observed during hypoxia can lead to a
lower threshold for firing action potentials as well as a more robust
firing response in the form of an increased frequency of spiking
(Tombaugh and Somjen 1996; Werkman et al.
1997
). Second, the increase in the persistent sodium current
during hypoxia would likely cause a significant shift in the
excitability potential of the cell due to the increase in membrane
conductance from the inward sodium current flow (Crill
1996
; Taylor 1993
). Thus these changes in the
sodium currents during hypoxia are most likely responsible for the
increased firing frequency and depolarization observed in caudal
hypothalamic neurons during hypoxia.
Neurons in the caudal hypothalamus have significant control over
cardiovascular and respiratory function. Stimulation of this area leads
to increases in respiratory rate, tidal volume, blood pressure, and
heart rate (Waldrop and Porter 1995). Although this region is not absolutely essential for basic cardiorespiratory function, it is believed that the primary function of neurons in the
caudal hypothalamus is to modulate cardiorespiratory function during
various physiological conditions (Horn and Waldrop
1998
). During periods of increased metabolic demand such as
exercise and hypoxia, caudal hypothalamic neurons are activated and,
via synapses on brain stem sympathetic and respiratory neurons,
contribute to the increases in ventilation and perfusion necessary
during these states (Horn and Waldrop 1997
; Ryan
and Waldrop 1995
; Vertes and Crane 1996
;
Waldrop and Porter 1995
; Waldrop et al.
1996
).
Recent work has focused on the specific manner in which neurons in the
caudal hypothalamus can react to changes in oxygen tension and
subsequently modulate respiratory output. This work was started 35 yr
ago in the rabbit with the demonstration of an increased firing
frequency during hypoxia in caudal hypothalamic neurons (Cross
and Silver 1963). More recent studies in the cat have shown
that a significant proportion of neurons in the caudal hypothalamus
increase their firing frequency during systemic hypoxia (Dillon
and Waldrop 1993
). In addition, the increase in firing rate
during hypoxia was maintained following the abolition of peripheral
chemoreceptor input. Similar results were found in the rat with the
added finding that a large number of these cells display rhythms
correlated to either the sympathetic or respiratory cycles and also
sent projections to midbrain and medullary cardiorespiratory centers
(Barman 1990
; Ryan and Waldrop 1995
).
When studied in an isolated in vitro brain slice preparation, a large
majority of rat caudal hypothalamic neurons are activated during
hypoxia that persists even during complete synaptic blockade
(Dillon and Waldrop 1992
). Therefore these neurons
appear to have the ability to directly respond to changes in oxygen
tension and likely transduce this response into an alteration in
physiological function via connections with the respiratory control
centers in the brain stem.
When analyzed using voltage-clamp techniques, caudal hypothalamic
neurons responded to hypoxia with a sustained inward current and
increase in membrane conductance (Horn et al. 1999).
This study showed that the inward current response is unaffected by calcium channel blockers but is almost completely abolished with the
sodium channel blocker, tetrodotoxin. Furthermore the increased membrane conductance is greatly reduced when extracellular sodium is
eliminated adding to the evidence of a sodium current mediating the
membrane response to hypoxia in these cells.
The present work significantly broadens our understanding of how sodium
currents are affected during hypoxia in caudal hypothalamic neurons.
The amplitude of the fast-inactivating sodium current in these neurons
was significantly increased during hypoxia. One explanation for this
result is that oxygen deprivation directly causes a conformational
change in the sodium channel that alters the conductance through this
channel. Alterations due to hypoxia in the amplitude of
fast-inactivating sodium current have been observed before in rat
hippocampal and human neocortical neurons; however, the alteration
observed in these studies was a decrease in current amplitude
(Cummins et al. 1993; O'Reilly et al.
1997
).
Because a wide variety of intracellular signaling molecules are known
to modulate sodium channel activity in mammalian neurons, another
possible mechanism of the change in current during hypoxia is a change
in intracellular signaling molecules by the hypoxic stimulus
(Catterall 1999; Ma et al. 1994
;
Smith and Goldin 1992
, 1997
). An
example of this type of modulation was shown in hippocampal neurons
where the hypoxic-induced decrease in sodium current amplitude was
blocked by protein kinase C (PKC) inhibitors (O'Reilly et al.
1997
). Because the amplitude of the fast-inactivating currents in caudal hypothalamic neurons was increased during hypoxia,
stimulation of PKC is unlikely to be responsible for the alteration
observed. In fact, one argument for the results observed in this study
would be that PKC is decreased during hypoxia due to a decrease in
aerobic metabolism that leads to a disinhibition of the
fast-inactivating currents. This is unlikely, however, since several
studies have shown that the activity of PKC is increased in neurons or
neuronal-like cells by short periods of hypoxia or ischemia
(Cardell and Wieloch 1993
; Gozal et al.
1998
; Kobayashi and Millhorn 1999
;
Wieloch et al. 1991
, 1993
).
Since hypoxia has been shown to decrease intracellular pH, the
modulation of sodium current during hypoxia may be explained by an
indirect effect of hydrogen ion concentration on channel function
(Fujiwara et al. 1992). In fact, the biophysical
properties of several ion channels and the activity of central neurons
are known to be pH dependent (Chesler 1990
; Dean
et al. 1989
; Lehmenkuhler et al. 1989
;
Liu et al. 1999
). Intracellular acidification, however, has been shown to decrease the function of fast-inactivating sodium currents in rat hippocampal neurons, suggesting a different mechanism for sodium current enhancement during hypoxia in caudal hypothalamic neurons (Tombaugh and Somjen 1996
). Therefore a
plausible mechanism for the hypoxic increase in the amplitude of this
current is a direct effect of a lack of oxygen on the channel, although
more substantial evidence through the use of single-channel recordings is needed.
Taken in conjunction with the hyperpolarized shift in the activation
curve for these neurons during hypoxia, the increase in
fast-inactivating sodium current amplitude would most likely cause an
enhancement of excitability. Since the main effects of hypoxia on
caudal hypothalamic neurons are a depolarization and increased firing
frequency, the modifications of the fast-inactivating sodium channels
observed in the present study can partially explain these responses.
The increase in amplitude of this sodium current indicates either a
change in channel conductance or gating properties leading to an
enhancement of excitability (Tombaugh and Somjen 1996).
In addition, the hyperpolarized activation curve would lower the
threshold for activation of these neurons during hypoxia leading to a
greater ability to spike (Werkman et al. 1997
). In contrast, the hyperpolarized inactivation curve during hypoxia could
lead to a decrease in excitation because the inactivation persists
longer into the repolarization phase and thus limits the ability to
increase firing frequency (Werkman et al. 1997
). However, the hyperpolarized inactivation curve could also allow the
neuron to repolarize more quickly due to the quicker initiation of
inactivation and thus lead to a shorter action potential duration and
subsequent increase in firing frequency. To differentiate between these
two possibilities, a detailed analysis of the effect of hypoxia on the
time course of inactivation recovery is needed.
In addition to the fast-inactivating sodium current, the
noninactivating or persistent sodium current was significantly enhanced by hypoxia in these neurons. The persistent sodium current can be
either TTX-sensitive or resistant and is 1-3% the amplitude of the
fast-inactivating current with similar voltage sensitivity (Bezanilla and Armstrong 1977; Chandler and Meves
1970
; Cummins and Waxman 1997
; Parri and
Crunelli 1998
). Single-channel analyses have added to the
growing evidence that these currents are a variant in the gating
kinetics of fast-inactivating sodium channels and not a distinct
protein channel (Alzheimer et al. 1993
; Keynes and Elinder 1998
). One of the primary roles postulated for this current in central neurons is to increase the firing rate of neurons through a sustained sub-threshold current (Taylor 1993
).
This can occur with such small currents because at the more
hyperpolarized level of activation in this current, the overall
impedance of the cell is high thereby allowing small currents to cause
depolarizations significant enough to reach the action potential
threshold (Crill 1996
). In addition, this current is
important in sustaining rhythmic firing behavior in a number of
neuronal types (Alonso and Llinas 1989
; D'Angelo
et al. 1998
). Recent single-channel work has shown that the
persistent sodium current in rat ventricular myocytes is enhanced
during a short period of hypoxia, which is due to an increase in the
open probability of the channel (Ju et al. 1996
).
Similar increases in the persistent sodium current during hypoxia were
found in rostral ventrolateral medullary neurons in the rat
(Kawai et al. 1999
). In these cells, which are
stimulated during hypoxia, both a decrease in molecular oxygen as well
as treatment with cyanide had similar enhancing effects on the
persistent sodium current. In contrast, cyanide caused a decrease in
the persistent sodium current in rat CA1 hippocampal neurons, which primarily respond to hypoxia with a hyperpolarization
(Hammarstrom and Gauge 1998
). Thus the effect of hypoxia
on this current directly correlates with the membrane response to
hypoxia in several cell types.
In conclusion, a brief period of hypoxia causes an increase in the amplitude of the fast-inactivating and persistent sodium currents in acutely dissociated rat caudal hypothalamic neurons. Furthermore the activation and inactivation curves for the fast-inactivating sodium current were significantly hyperpolarized during hypoxia in these cells. These results suggest that sodium currents are primarily responsible for the depolarization and increased firing frequency response observed in these cells during hypoxia and at least in part comprise the cellular mechanism allowing these neurons to modulate respiratory function during hypoxia.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. Gabriel G. Haddad for advice on the acute neuronal dissociation protocol and Dr. Phillip Best for a critical review of the manuscript.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-06296 and American Heart Association-Illinois Affiliate Grant-in-Aid to T. G. Waldrop and American Heart Association-Illinois Affiliate Grant 9704427A to E. M. Horn.
Present address of E. M. Horn: Barrow Neurological Institute, Division of Neurosurgery, 350 W. Thomas Ave., Phoenix, AZ 85013.
![]() |
FOOTNOTES |
---|
Address for reprint requests: T. G. Waldrop, Dept. of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, MC-114, 407 S. Goodwin Ave., Urbana, IL 61801 (E-mail: twaldrop{at}uiuc.edu).
Received 22 March 2000; accepted in final form 28 July 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|