1Department of Pediatrics (Section of Respiratory Medicine) and 2Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gu, Xiang Q.,
Hang Yao, and
Gabriel G. Haddad.
Effect of Extracellular HCO3 on Na+
Channel Characteristics in Hippocampal CA1 Neurons.
J. Neurophysiol. 84: 2477-2483, 2000.
The effect of
HCO3
/CO2 on
membrane properties of isolated hippocampal CA1 neurons was studied
with the use of the whole cell configuration of the patch-clamp
technique. Neurons were acutely dissociated from 21- to 30-day-old
mice. In the current-clamp mode,
HCO3
/CO2
significantly hyperpolarized CA1 neurons by more than 10 mV and
decreased their input resistance. In addition, the overall excitability
of these neurons was lower in the presence of
HCO3
/CO2 than in
HEPES. Spontaneous and evoked action potential firing frequency was
lower in the presence of
HCO3
/CO2 than in
its absence. In the voltage-clamp mode, both activation and
steady-state inactivation of a fast Na+ current
were shifted in the hyperpolarized direction in such a way that the
window currents were smaller in
HCO3
/CO2 than in
HEPES. Recovery from inactivation and deactivation from the open state
of the fast Na+ current was slower in
HCO3
/CO2 than in
HEPES. We conclude that
HCO3
/CO2
decreases the intrinsic excitability of CA1 neurons by altering not
only the passive properties of the neuronal membranes but also by
changing several characteristics of the fast Na+
current, including activation and inactivation kinetics as well as the
recovery from inactivation and deactivation.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most electrophysiologists use
HCO3/CO2 in the
extracellular solution in slice preparations but use HEPES saline in
whole cell experiments. Although changing solutions from HEPES to
HCO3
/CO2 or vice
versa is used very frequently when the role of certain membrane
proteins such as those regulating Na+ or
H+ intracellularly is investigated, the
electrophysiologic alterations that occur in neurons under these two
conditions are ill defined. For example, although some data are
available (Church 1992
; Cowan and Martin
1995
, 1996
; Stea and Nurse 1991
),
there are still gaps in our knowledge as to how excitability is altered
in neurons with
HCO3
/CO2 and, if
so, what is the basis for the alteration. Since the presence of
extracellular
HCO3
/CO2 can
activate electrogenic transporters (Brune et al. 1994
; de Hurtado et al. 1995
; Deitmer and Schlue
1989
) and can change pHi (Bevensee
and Boron 1998
; Bevensee et al. 1996
,
1997
; Bonnet et al. 1998
;
Saarikoski et al. 1997
; Yao et al. 1999
),
a number of electrophysiologic changes could be expected. We therefore undertook this study to investigate the neuronal responses to the
changes imposed in the extracellular milieu and focused on the
potential mechanisms that underlie excitability under various conditions. Because we have used CA1 hippocampal neurons extensively in
our previous studies, we chose to investigate how these particular neurons respond to the change in the extracellular fluid.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Solutions
For the current-clamp experiments, the external HEPES solution
bathing neurons contained (in mM) 130 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES,
and 10 glucose, adjusted to pH 7.4 with NaOH. The
HCO3/CO2
solution contained (in mM) 125 NaCl, 3.1 KCl, 1.25 NaH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose, bubbled with 5% of
CO2 and 95% of O2. The
internal pipette solution contained (in mM) 138 KCl, 0.2 CaCl2, 1 MgCl2, 10 HEPES
(Na+ salt), and 10 EGTA, adjusted to pH 7.4 with
tris. The external HEPES and
HCO3
/CO2
solutions for voltage-clamp experiments bathing neurons contained the
same salts as in the current-clamp experiments, except for 10 mM TEA, 5 mM 4-aminopyridine (4-AP), and 0.1 mM CdCl2 that replaced equal molar concentrations of NaCl. The internal pipette solution for the voltage-clamp experiments was also similar to the
internal solution for the current-clamp experiments, except for CsF or
CsCl instead of KCl. The HEPES-buffered solutions for the enzymatic
preparation and trituration of the CA1 cells contained (in mM) 125 NaCl, 3 KCl, 1.2 MgSO4, 1.25 NaH2PO4, 30 HEPES, and 10 glucose. Osmolarity of all solutions was adjusted to 290 mOsm. All
chemicals were purchased from Sigma.
Recording criteria
MORPHOLOGIC CRITERIA.
CA1 cells were used if they had a smooth surface, a three-dimensional
contour, and pyramidal shape. CA1 cells studied were from 21- to
30-day-old mice. Cells were not considered for recording when they had
flat or grainy surfaces. Similar criteria have been used by us
(Cummins et al. 1994) and others (Hamill et al.
1981
) on freshly tritrurated neurons.
ELECTROPHYSIOLOGIC CRITERIA.
1) The seal resistance was measured using Ohm's law
immediately after patching each cell to assess the quality of the seal. Neurons were considered for recording if the seal resistance was >5
G. 2) Only neurons with a holding current of <0.1 nA
(command potential
100 mV) on establishment of the whole cell
configuration were used in the study. 3) Series resistance
was <10 M
in neurons studied. When measured in different solutions,
series resistances were 6.83 ± 0.75 (mean ± SE,
n = 12) in HEPES and 5.80 ± 0.92 (n = 7) in
HCO3
/CO2
solution. The series resistances were compensated at 80-85% level
with the Axopatch 1C amplifier (Axon Instruments). Under these
conditions, the error caused by uncompensated series resistances was
<0.5 mV, since the cells used had a mean
INa+ of <2.5 nA. To obtain
adequate voltage clamp and minimize the space-clamp problem, only small
neurons with short processes were used in
INa+ measurement. In
addition, only cells with current-voltage (I-V) 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
(O'Reilly et al. 1997
).
Electrophysiologic recording
All recordings were performed at room temperature (22-24°C).
Electrodes were pulled on a Flaming/Brown micropipette puller (Model
P-87, Sutter Instrument) from filamented borosilicate capillary glass
(1.2 mm OD, 0.69 mm ID, World Precision Instruments). The electrodes
were fire-polished, and resistances were 2-5 M measured in the
above solutions. Action potentials (AP) were recorded in the
current-clamp mode, and AP duration was measured at half voltage between AP threshold and AP peak. Rm
was measured at
70 mV as a slope of the current trace evoked by a
ramp voltage from
160 to 100 mV in the voltage-clamp mode.
Least-square regression analysis for 100 data points was performed to
derive a relationship between voltage and current. Alternatively, in
current-clamp mode, and at
75 mV holding potential, a 0.01 nA
hyperpolarizing current was given and the voltage was collected to
calculate the Rm.
Rms derived from the two methods were
similar. Vm was measured in the
current-clamp mode with no holding current. All values reported are
represented in means ± SE.
Preparation of CA1 cells
Mice, 21-30 days old (SJL from the Jackson Laboratory) were used, and their hippocampi were removed and sliced into 7-10 transverse sections of 400 µm thick. The slices were immediately transferred to a container with 25 ml of fresh, oxygenated, and slightly stirred HEPES buffer at room temperature. After 30 min of exposure to trypsin (0.08%) and 20 min of protease (0.05%) digestion, the slices were washed several times with HEPES buffer and left in oxygenated solution. The CA1 region was then dissected out and triturated in a small volume (0.25 ml) of HEPES buffer. These studies have been approved by the Yale Animal Care and Use Committee.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Morphology, passive and active properties
Acutely dissociated mouse CA1 neurons were often in an isolated form (Fig. 1A) and had varied morphology. Most cells inspected had a neuronal appearance: they had a three-dimensional form and cellular processes and were elliptical, rounded, or pyramidal in shape. For consistency, we studied only pyramidal neurons.
|
We first studied a number of properties at rest, in the nominal absence
of HCO3/CO2,
i.e., in HEPES or in the presence of
HCO3
/CO2-buffered
solutions. Most neurons studied were rather small, and the average
whole cell capacitance in the absence of
HCO3
/CO2 was
6.0 ± 0.5 pF (mean ± SE, n = 22). We also
examined in detail a number of other properties. For example, with
depolarizing currents of 0.1 nA and a holding voltage of
75 mV, mean
AP threshold was
54.1 ± 2.1 mV, mean AP amplitude was 83.5 ± 4.7 mV, and mean AP duration was 1.9 ± 0.2 ms
(n = 21). Mean Vm in
these CA1 neurons was
42 ± 2 mV (n = 19; Fig.
1B), and the mean input resistance (Rm) was 696 ± 88 M
(n = 17; Fig. 1C).
HCO3/CO2
solutions altered a number of these properties. Whole cell capacitance
was 8.0 ± 0.8 pF, and this was significantly larger than in HEPES
(n = 12, P = 0.02). With depolarizing
currents, mean AP threshold was
54.1 ± 2.2 mV
(n = 8), with an average AP amplitude of 77.4 ± 8.7 mV (n = 8) and a mean AP duration of 1.5 ± 0.1 ms (n = 8).
HCO3
/CO2
significantly hyperpolarized CA1 neurons (mean
Vm =
53 ± 4 mV,
n = 18, P = 0.004; Fig. 1B)
and decreased Rm (mean
Rm = 385 ± 46 M
,
n = 21, P = 0.01; Fig. 1C).
Thus HCO3
/CO2
solutions increased cell capacitance, rendered
Vm more negative, and decreased
Rm.
To test whether an electrogenic HCO3
mechanism was involved in hyperpolarizing the cells in the presence of
HCO3
/CO2, we
used DIDS to inhibit transporters that depend on
HCO3
. In the presence of
HCO3
/CO2, DIDS
(0.5 mM) caused 5.6 ± 1.0 mV (n = 5)
depolarization, and this depolarization was reversible when DIDS was removed.
Neuronal excitability in the presence and absence of
HCO3/CO2
SPONTANEOUS FIRING.
The effect of
HCO3/CO2 on
spontaneous firing was studied in detail in 12 CA1 neurons. Figure
2A shows a CA1 neuron firing APs at a relatively low rate in bicarbonate solution. When switched from HCO3
/CO2 to
HEPES solution, the same neuron fired APs at a much higher rate. When
the perfusing solution was changed back to
HCO3
/CO2
solution, the neuron fired at a lower rate again. Indeed, although CA1
neurons had an irregular pattern of firing, they had generally
spontaneous APs in HEPES. However, in all CA1 cells studied, they
either did not have any spontaneous APs in
HCO3
/CO2
solution, or their firing rate was much lower than that in HEPES
solution.
|
EVOKED FIRING OF APS.
Forty-two CA1 neurons were studied, and all fired APs when they were
held at 75 mV and given depolarizing currents in the current-clamp
mode. For example, in Fig. 2B, a 0.05 nA depolarizing current evoked nine APs in this particular neuron in the absence of
HCO3
/CO2. When
the solution was changed to
HCO3
/CO2, two
APs were evoked in this same neuron. Switching back to HEPES reversed
the inhibition, and the number of evoked APs increased to four. In some
neurons, firing was totally eliminated when exposed to
HCO3
/CO2. APs in
all neurons reappeared with the removal of
HCO3
/CO2. On
average, the number of APs evoked by depolarizing currents of 0.1 nA
was reduced from 3.9 ± 0.9 (n = 21) in the
absence of HCO3
/CO2 to
1.6 ± 0.4 (n = 8) in
HCO3
/CO2
solution. Also, less current was needed to generate the same numbers of
APs in the absence than in the presence of
HCO3
/CO2. In
Fig. 2C, the rheobase was much smaller in the absence of
HCO3
/CO2 than in
the presence of
HCO3
/CO2:
61.7 ± 14.4 pA were needed to generate one AP in the absence of
HCO3
/CO2,
whereas 147.5 ± 35.5 pA (n = 12, P = 0.02) were needed in HCO3
/CO2.
Similarly, to evoke two, three, four, or more APs, the currents used
were always smaller in the absence of
HCO3
/CO2 than in
the presence of
HCO3
/CO2.
Clearly, one explanation for the decrease in excitability seen in
neurons in
HCO3
/CO2 is
related to the hyperpolarization and decrease in
Rm. Note, however, that in Fig.
2B, AP generation in the presence or absence of
HCO3
/CO2 was
induced from the same membrane potential, i.e.,
75 mV. Therefore
during evoked stimulation, the difference in excitability observed in
HCO3
/CO2 and in
HEPES cannot be attributed to differences in
Vm since we controlled that variable.
The drop in Rm with
HCO3
/CO2 could,
by itself, contribute to the decrease in excitability.
Fast Na+ current
Since evoked firing of CA1 neurons was lower in the presence than
in the absence of
HCO3/CO2 in
spite of the fact that Vm was held at
the same level, we raised the question as to whether the presence of
HCO3
/CO2
affected other membrane properties. For example,
Na+ channel properties and kinetics would be
important to examine and could have been altered. If this were the
case, we would suspect that
HCO3
/CO2 would
change the properties of Na+ channels in such a
way to make CA1 neurons less excitable.
When we held CA1 neurons at 130 mV, depolarizing pulses to
20 mV
evoked an inward current that reached a peak in less than a millisecond
and decayed subsequently to zero current (Fig.
3A). This inward current was
carried by Na+ since TTX (1 µM) blocked the
current almost totally (data not shown). Based on its voltage
dependency, characteristics of fast activation and TTX sensitivity, we
considered this as a voltage-sensitive fast Na+
current. Switching solutions from the nominal absence of
HCO3
/CO2 to one
containing
HCO3
/CO2 changed
neither the size of the fast Na+ current of CA1
neurons nor the fast Na+ current density (peak
current/capacitance; data not shown).
|
NA+ ACTIVATION CHARACTERISTICS.
With CA1 neurons held at 130 mV, depolarizing voltages were given
from
70 to 80 mV for 48 ms at increments of 10 mV. In the absence and
presence of
HCO3
/CO2, the
threshold for Na+ current activation was about
60 mV (Fig. 3B,
). The Na+
currents increased in amplitude with depolarizing voltages and reached
a peak at about
20 mV in HEPES and
30 mV in
HCO3
/CO2. The
normalized conductance, plotted against
Vm, showed a much more hyperpolarized
midpoint
(m
,1/2) in
the presence (
50 mV) than in the absence (
39 mV) of
HCO3
/CO2
(n = 6, Fig. 3C).
STEADY-STATE INACTIVATION CHARACTERISTICS OF
NA+ CURRENTS.
Steady-state inactivation of Na+ currents was
studied with current traces obtained using a prepulse potential from
130 to
20 mV for 502 ms and then stepping
Vm to
20 mV in the absence and
presence of
HCO3
/CO2. Figure
4, A and B, shows
an example of current traces from the same cell under voltage clamp.
When the normalized current (I/Imax) was plotted
against the prepulse potential, the relationship could be fitted with a
Boltzmann equation. The midpoints of the steady-state inactivation
curves
(h
,1/2) were
63 and
83 mV in the absence and presence of
HCO3
/CO2,
respectively. Note that the maximum currents were obtained from
prepulse of
130 to
90 mV (n = 6). The amplitude of
the Na+ currents was decreased with more
depolarized prepulse potentials, ranging from
90 to
40 mV. At
voltages more depolarized than
40 mV, little or no
Na+ current was generated in either solutions.
Changing solution from HEPES to
HCO3
/CO2,
shifted h
,1/2
in the hyperpolarized direction by about 20 mV. The
h
1/2 was
63 and
83 mV in the absence and presence of
HCO3
/CO2,
respectively (n = 6). As can be seen from the above
results, since the activation curve is shifted by about 11 mV and the
steady-state inactivation by about 20 mV in the hyperpolarized
direction when the extracellular solution is changed to
HCO3
/CO2, the
window currents became narrower in
HCO3
/CO2 than in
HEPES solution (Fig. 5).
|
|
NA+ CURRENT RECOVERY FROM INACTIVATION.
We studied recovery from inactivation of the fast
Na+ current by using a two-pulse protocol. Rate
of recovery from inactivation of CA1 neurons was lower in
HCO3/CO2
solution than that in the absence of
HCO3
/CO2 (Fig.
6). For example, if two pulses were 5.4 ms apart, the ratios of the second peak Na+
current over the first were 0.62 ± 0.09 and 0.39 ± 0.03 (n = 5, P = 0.02) in the absence and in
the presence of
HCO3
/CO2,
respectively. The time constants (
) for recovery were 2.44 ± 0.56 and 5.34 ± 0.58 (n = 5, P = 0.03) in HEPES and
HCO3
/CO2
solutions.
|
DEACTIVATION CHARACTERISTICS OF NA+
CURRENTS.
We also examined the effect of
HCO3/CO2 on the
transition from the open to the resting closed state. We held CA1
neurons at
100 mV, depolarized them for 1 ms to
10 mV, and
repolarized to
70 or
100 mV. In HEPES and
HCO3
/CO2, the
repolarizing voltage to
100 mV evoked inward currents that decayed to
zero rapidly. When we repolarized neurons to
70 mV, the decay of the
evoked currents was much slower. The averaged decay constants at
100
mV were 0.18 ± 0.02 (n = 4) in HEPES solution and
0.23 ± 0.05 (n = 4) in
HCO3
/CO2. At
70 mV, the averaged decay constant was much slower (0.88 ± 0.20, n = 4) in the presence of
HCO3
/CO2 than in
HEPES (0.29 ± 0.06, n = 4; P = 0.03). Thus
HCO3
/CO2
significantly increased the time constant of deactivation of the fast
Na+ channel in CA1 neurons when they were
repolarized to
70 mV after a brief depolarization.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although there have been a few studies detailing the effect of a
change in intracellular pH on the activity of exchangers and some of
the neuronal properties (Bevensee and Boron 1998; Bevensee et al. 1996
, 1997
; Bonnet
et al. 1998
; Brooks and Bachelard 1992
;
Church 1992
; Cowan and Martin 1995
;
Dart and Vaughan-Jones 1992
; de Hurtado et al.
1995
; Deitmer 1992
; Gaillard and Dupont 1990
; Saarikoski et al. 1997
; Yao et al.
1999
), there has not been a comprehensive investigation on the
role of HCO3
/CO2
bathing neurons in neuronal excitability and the basis for it. In this
work we have made two observations, both of which contribute to the
decrease in neuronal excitability with
HCO3
/CO2.
Although the extracellular pH is kept at the same level, the presence
of HCO3
/CO2 in
the extracellular milieu alters profoundly 1) the passive neuronal membrane properties and 2) the
Na+ channel kinetics.
MEMBRANE PROPERTIES AND NEURONAL EXCITABILITY IN THE PRESENCE OF
HCO3/CO2.
It is clear from our studies that excitability decreases in the
presence of
HCO3
/CO2. The
rheobase in these CA1 neurons increased, and, with the same amount of
current injected, the number of action potentials fired with
HCO3
/CO2 was
lower than that with HEPES. One major reason for this decrease in
excitability with
HCO3
/CO2 is the
decrease in Vm (more negative) and
decrease in Rm. With respect to
Vm, we believe that it is higher
because of the effect of
HCO3
/CO2 on
possibly a number of membrane proteins, the activity of which depends
on the presence of HCO3
. Although
intracellular pH (pHi) increases with
HCO3
/CO2
(Yao et al. 1999
) in these CA1 neurons, clearly, the
difference in proton concentrations between inside and outside of the
cell cannot explain the change in Vm
with HCO3
/CO2 by
the Nernst equation. Hence other factors are important, and we
hypothesized that it is the activation of one or more electrogenic, HCO3
-dependent transporters that are
at the basis of the change in Vm.
Indeed, we have shown that DIDS, a blocker of
HCO3
-dependent transporters,
depolarized these CA1 cells. Hence, we have evidence that
HCO3
transporters play a role in the
electrogenicity of these cells. Although it has been assumed that this
co-transporter exists only in glia, we have recently shown that this
membrane protein is also present in neurons and in the neurons we have
studied in this work (Schmitt et al. 2000
). While it is
possible that
HCO3
/CO2
activates more than one membrane protein, the net result observed can
be explained only by implicating the activation of at least one such
electrogenic membrane protein. It is important to keep in mind,
however, that HCO3
can activate both
acid extruders and loaders, with some electrogenic and others not. It
is clearly possible that the activation of any number of these
transporters could increase the conductance of these neurons, as we
have observed in our studies. Furthermore, it is worth mentioning that
the effect of
HCO3
/CO2 on
Vm depends clearly on the membrane
protein endowment of the neuron; therefore it is likely that the effect
of HCO3
/CO2
varies from one cell type to another.
NA+ CHANNELS AND NEURONAL EXCITABILITY IN
THE PRESENCE OF HCO3/CO2.
In this study, although HCO3
increased Vm,
our electrophysiologic pulse paradigm clamped cells at the same
potential, irrespective of the bathing solution. In spite of clamping
Vm, cells were still less excitable in
HCO3
/CO2. The
increase in conductance could still contribute to the decreased
neuronal excitability, but we found in this work that Na+ channel kinetics are altered in such a way to
lessen excitability.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by National Institutes of Health Grants P01-HD-32573 and NS-35918 to G. G. Haddad.
![]() |
FOOTNOTES |
---|
Address for reprint requests: G. G. Haddad, Dept. of Pediatrics, Section of Respiratory Medicine, Rm. 508, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510 (E-mail: gabriel.haddad{at}yale.edu).
Received 20 March 2000; accepted in final form 28 July 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|