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The Journal of Neuroscience, July 15, 1998, 18(14):5212-5224
Response of Thalamocortical Neurons to Hypoxia: A Whole-Cell
Patch-Clamp Study
Gül
Erdemli and
Vincenzo
Crunelli
Physiology Unit, School of Molecular and Medical Biosciences,
University of Wales Cardiff, Cardiff, CF1 3US, United Kingdom
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ABSTRACT |
The effect of hypoxia (3-4 min of 95% N2, 5%
CO2) on thalamocortical (TC) neurons was
investigated using the whole-cell patch-clamp technique in rat dorsal
lateral geniculate nucleus slices kept submerged at 32°C. The
predominant feature of the response of TC neurons to hypoxia was an
increase in input conductance ( GN = 117 ± 15%, n = 33) that was accompanied by
an inward shift in baseline holding current
(IBH) at  65 and 57 mV
(IBH = 45 ± 6 pA,
n = 18, and 25 ± 8 pA,
n = 33, respectively) but not at 40 mV. The
hypoxia-induced increase in GN (as well as
the shift in IBH) was abolished by
procedures that are known to block
Ih, i.e., bath application of
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride (100-300 µM) (GN = 5 ± 13%, n = 11) and CsCl (2-3
mM) (GN = 16 ± 16%,
n = 5), or low
[Na+]o
(GN = 10 ± 10%,
n = 5), whereas bath application of
BaCl2 (0.1-2.0 mM) had no significant effect
(GN = 128 ± 14%,
n = 8). The hypoxic response was also abolished in
low [Ca+2]o
(GN = 25 ± 16%,
IBH = 6 ± 8 pA,
n = 13), but was unaffected by recording with
electrodes containing EGTA (10 mM), BAPTA (10-30 mM), Cs+, or Cl , as
well as in the presence of external tetraethylammonium and 4-aminopyridine. Furthermore, preincubation of the slices with botulinum toxin A (100 nM), which is known to reduce
Ca2+-dependent transmitter release, blocked the
hypoxic response (GN = 3 ± 15%,
IBH = 10 ± 5 pA,
n = 4).
We suggest that a positive shift in the voltage-dependence of
Ih and a change in its activation kinetics,
which transforms it into a fast activating current, may be responsible
for the hypoxia-induced changes in GN and
IBH, probably via an increase in
Ca+2-dependent transmitter release.
Key words:
hypoxia; Ih; inward
rectification; cesium; ZD 7288; dorsal lateral geniculate nucleus; botulinum toxin; transmitter release
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INTRODUCTION |
A common feature of many mammalian
neurons is their sensitivity to oxygen deprivation, although different
types of neurons, even within the same brain region, show large
variations in their sensitivity to hypoxia/ischemia (Hochachka et al.,
1993 ; Krnjevic, 1993; Martin et al., 1997). Brief periods of hypoxia,
for instance, cause a total, but apparently fully reversible, loss of
synaptic transmission in the hippocampus (Leblond and Krnjevic, 1989;
Krnjevic, 1993), which may be responsible for the rapid disruption of
higher brain functions in the absence of oxygen. The prominent feature of the hypoxic response in hippocampal neurons is an increase in input
conductance (GN) associated with an
outward shift in baseline holding current
(IBH). This effect is believed to be caused mainly by an enhanced K+ conductance that may
serve as a protective mechanism to decrease or delay excitotoxicity
(Fujiwara et al., 1987; Krnjevic and Leblond, 1989; Leblond and
Krnjevic, 1989). On the other hand, brainstem neurons, which subserve
autonomic functions, are slightly depolarized in response to hypoxia,
so that they maintain cardiovascular functions during oxygen
deprivation (Haddad and Donelly, 1990; Cowan and Martin, 1992; Haddad
and Jiang, 1993; Nolan and Waldrop, 1996).
The thalamus shows a marked sensitivity to ischemic insults
(Szelies et al., 1991; Steinke et al., 1992), and in
situ immunohistochemical studies have revealed that both ventral
and dorsal thalamic nuclei (including the ventral posterolateral, the
ventral posteromedial, and the medial and dorsal lateral geniculate
nucleus) are highly sensitive to ischemic challenges. Indeed, these
nuclei, together with the primary sensory cortex and the basal ganglia,
appear to be part of a system-preferential, topographically organized brain injury that contributes to a selective vulnerability to ischemia,
particularly in the newborn (Martin et al., 1997). Although these
thalamic nuclei have recently been the subject of many
electrophysiological studies because of their central role in various
physiological functions and in a number of neurological disorders
(Jones, 1985; Steriade and Llinas, 1988; Steriade et al., 1993;
Williams et al., 1996, 1997; McCormick and Bal, 1997; Turner et al.,
1997), little is known about the electrical behavior of single
thalamocortical (TC) neurons in response to hypoxia.
In the present experiments, we have investigated the effects of brief
periods of hypoxia on TC neurons of the dorsal lateral geniculate
nucleus (dLGN) maintained in slices using the whole-cell patch-clamp
technique. Our results suggest that activation of the
hyperpolarization-activated inward current,
Ih, brought about by a positive shift in
its voltage-dependence and changes in its kinetics, is the major factor
responsible for the response of TC neurons to hypoxia.
Preliminary reports of some of these results have been published
previously (Crunelli and Erdemli, 1997; Erdemli and Crunelli, 1998).
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MATERIALS AND METHODS |
Slice preparation and recording solutions. For the
preparation of dLGN slices, rats (Wistar, 100-150 gm) were decapitated under full anesthesia with halothane. The brain was quickly removed and
placed in an ice-cold oxygenated saline (Williams et al., 1996, 1997).
A block of brain tissue containing the thalamus was dissected out, and
400-µm-thick dLGN slices were cut in a plane parallel to the optic
tract using a vibroslice (Campden Instruments). Slices were then kept
for at least 1 hr at room temperature in the standard artificial CSF
(ACSF) containing (in mM): NaCl 134, KCl 2, KH2PO4 1.25, Mg2SO4 1, CaCl2 2, NaHCO3 16, and glucose 10, and were
aerated continuously with carbogen (95% O2, 5%
CO2, pH 7.3).
Before the start of the electrical recording, a slice was transferred
to a submerged chamber, where both the ACSF and the aerating gas were
warmed to 32 ± 1°C. The patch electrodes (2.5-3 µm tip
diameter) were prepared from 1.5 mm outer diameter borosilicate glass
(Clark Electromedical Instruments, Pangbourne, UK) and filled with
solution containing (in mM): KMeSO4 118, KCl
18, HEPES 10, EGTA 1 or 10, CaCl2 0.1 or 1, Mg-ATP 2, Na-GTP, 0.3, and NaCl 8. When it was necessary, KMeSO4 was
replaced by KCl and in some other experiments KMeSO4 and
KCl were replaced by cesium gluconate and CsCl, respectively. In a few
experiments, CsF (118 mM) was used instead of cesium
gluconate, and because the results obtained with these two internal
solutions were similar, data were pooled together. In some experiments
EGTA was replaced with BAPTA (10 or 30 mM). The pH was
always adjusted to 7.2 with KOH or CsOH. The osmolarity of the internal
solution (measured with a micro-osmometer, Viescor Inc.) was kept in
the range of 310-320 mOsm by reducing [KMeSO4] as
needed.
To minimize the indirect effects of synaptic transmission, slices were
always perfused with ACSF containing kynurenic acid (KYN, 1 mM), picrotoxin (PIC, 100 µM), and
tetrodotoxin (1 µM) (Ben-Ari, 1990a). In some experiments
PIC was replaced by ()-bicuculline methiodide (30 µM),
and DL-2-amino-5-phosphonopentoic acid (100 µM) (Tocris Neuramine) and
6-cyano-7-nitroquinoxaline-2,3-dione (20 µM) (Tocris
Neuramine) were used instead of KYN. In recordings with
Cs+-filled electrodes, tetraethylammonium chloride
(TEA, 10-20 mM) was also added to the perfusion medium.
For low Na+ (16 or 100 mM) solution,
Na+ was replaced either with
N-methyl-D-glucamine (134 mM,
tittered with HCl to pH 7.3) or TEA (50 mM), and
Ca2+ currents were blocked by a low
Ca2+ (0.5 mM) to high
Mg2+ (8-10 mM) solution containing
CdCl2 (300 µM) and NiCl2 (1 mM) (Crunelli et al., 1989; Guyon and Leresche, 1995). The
following agents were also tested by bath application: 4-aminopyridine
(4-AP, 0.1 mM), botulinum toxin A (100 nM, from
a stock solution in 0.2 M NaCl2 and 0.05 M sodium acetate), BaCl2 (0.1-2
mM), CsCl (2-3 mM),
4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium chloride) (ZD 7288, Zeneca, UK) (100-300 µM). Chemicals
were purchased from Sigma (Poole, UK), except where indicated.
Patch recording and data analysis. The patch electrodes had
an initial resistance of 2-3 M , and data from whole-cell recordings where the electrode series resistance increased to values >13 M
were discarded. All recordings were done "blind," and the criteria used to identify TC neurons included the presence of a relatively large
inward rectification, low-threshold Ca2+ potentials,
and strong outward rectification (Williams et al., 1996). In some
experiments, 0.5-1% biocytin was included in the intracellular patch
electrode solution. At the end of these recording sessions, the slices
were immediately fixed and then processed as described previously
(Williams et al., 1996).
The electrical signals were amplified by an Axopatch 1D (Axon
Instruments, Foster City, CA), and the data were analyzed with pClamp
(v6.1, Axon Instruments). Cells were clamped near resting membrane
potential (60 mV), and voltage-dependent currents were elicited by
1-sec-long hyperpolarizing and depolarizing voltage steps.
Current-voltage (I-V) relationships were
constructed using the instantaneous current evoked by hyperpolarizing
voltage steps, and the steady-state current was evoked by depolarizing
voltage steps. The GN was obtained from the
slope of the first-order regression lines fitted to the linear portion
of the I-V plots, in the region negative to the
holding potential.
The activation curve of Ih was constructed from
the amplitude of the inward relaxations calculated by subtracting the
instantaneous current from the steady-state current elicited by
hyperpolarizing voltage steps. Ih tail currents
were not used for this purpose, because the contributions of the
low-threshold Ca2+ current could not be eliminated
because of sensitivity of the hypoxic response to extracellular
Ca2+ (see Results). For the results presented in
Figure 4, the hypoxic current Idif (i.e., the
instantaneous current recorded during hypoxia minus the
instantaneous current recorded in control conditions) and
Ih were normalized to their respective maximal
amplitude and plotted against the step potential. The resulting data
were fitted with the Boltzmann equation of the form y = 1/[1 + e(V1/2 Vm)/k], where
Vm is the membrane potential,
V1/2 is the membrane potential at which
Idif or Ih is
half-activated, and k is the slope factor.
The effects of hypoxia were examined by using the method described by
Leblond and Krnjevic (1989). The periods of exposure to ACSF saturated
with 95% N2, 5% CO2 were 3-4 min
long, i.e., 1-2 min longer than in the experiments by Leblond and
Krnjevic (1989), in which slices were directly exposed to the aerating gases. The longer exposure used in the present study was to ensure that
a major decrease in oxygen was indeed achieved in the submerged slices.
Data were collected after the first 2 min of oxygen deprivation.
All quantitative data in the text and figures are expressed as
mean ± SEM, and their significance was assessed by Student's t test. For unpaired differences with unequal population
variances, the significance was estimated using the
d-statistic and Fisher-Behrens distribution (Campbell,
1989).
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RESULTS |
The effect of hypoxia were studied in a total of 106 TC neurons.
At a holding potential (VH) of
57.7 ± 0.8 mV, the initial resting GN
and the IBH of these 106 neurons were 5.8 ± 0.4 nS and 27 ± 11 pA, respectively.
Effects of hypoxia
In the 33 neurons that were recorded with electrodes containing
KMeSO4 and clamped at approximately 57 mV, a brief period of hypoxia caused a marked and consistent increase in
GN (GN = 117 ± 15%) (n = 33) and a small but significant change in
IBH (IBH = 25 ± 8 pA; p < 0.02 from the control
IBH=33 ± 16 pA; n = 33)
(Fig. 1A,B, Table
1). These effects were invariably
accompanied by a marked increase in the instantaneous current (101 ± 17%, n = 21), and a small increase in steady-state
current (21 ± 6%, n = 21) evoked by
hyperpolarizing voltage steps, resulting in a substantial reduction
(77 ± 4%, n = 21) of the amplitude of the inward
relaxations (Fig. 1A) (control amplitude: 294 ± 12 pA, n = 29; measured at 120 mV) attributable to
activation of Ih (McCormick and Pape, 1990a;
Soltesz et al., 1991; Pape, 1996). The inward shift in
IBH was more prominent and consistent in 18 of
these neurons clamped at 65 mV (IBH = 45 ± 6 pA; p < 0.001) (Table 1). On the other
hand, 13 hypoxic tests performed while the neurons were held at 40 mV
did not cause any significant change in IBH
(IBH = 5 ± 25 pA; n = 9), although the increase in GN
(GN = 111 ± 23%; n = 9) and the decrease in the inward relaxations (83 ± 12%;
n = 9) were still present. The reversal potential of
the hypoxia-evoked current(s), obtained from the point of intersection
of the instantaneous I-V plots in control and
during hypoxia, was 53.7 ± 3.2 mV (n = 25).
This value was not different from the reversal potential measured from
the intersection of the regression lines fitted to the linear portion
of the I-V plots (53.2 ± 4.7 mV;
n = 25).
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Figure 1.
Hypoxia causes an increase in
GN, an inward shift in
IBH, and a decrease in the amplitude
of inward relations in TC neurons. A1,
Voltage-clamp traces obtained with an electrode containing
KMeSO4 show the inward and outward currents evoked by
depolarizing and hyperpolarizing voltage steps before
(CONTROL), during (HYPOXIA), and 5 min
after hypoxia (RECOVERY). Note the marked
increase in instantaneous current and the small increase in
steady-state current evoked by the hyperpolarizing voltage steps during
hypoxia. A2, Continuous trace shows the inward
current activated during hypoxia. B1,
I-V plot obtained from the same neuron
as in A1 shows the substantial increase in
GN during hypoxia. In this and other
I-V plots in the following figures,
open circles, open squares, and
closed triangles represent data obtained before, during,
and after hypoxia, respectively. B2, Plot of the
difference current Idif (i.e., the
instantaneous current measured during hypoxia minus the
instantaneous current measured in control conditions) from the data
shown in B1. In this and the following figures,
Vh indicates the holding potential (for
further details, see Materials and Methods).
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The effect of hypoxia was reversible: 4-5 min after readmission of
oxygen, GN,
IBH, and the amplitude of the inward
relaxations were not significantly different from the corresponding
control values (9 ± 17%, n = 27; 13 ± 10%, n = 27; and 13 ± 11%, n = 21, respectively) (Figs. 1A,B,
2, 3; see
Figs. 5-8). In addition, the hypoxic challenge could be repeated at
intervals of 10-15 min without any obvious long-lasting effect. Thus,
in contrast to previous observations in other neuronal types (Reid et
al., 1984; Leblond and Krnjevic, 1989), TC neurons responded to hypoxia in a very consistent and reproducible manner.
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Figure 2.
The hypoxic response of TC neurons is not blocked
by intracellular Cs+. Whole-cell recording with a
patch electrode containing cesium gluconate. A, Examples
of currents evoked by hyperpolarizing voltage steps before
(CONTROL), during (HYPOXIA), and 4 min
after hypoxia (RECOVERY). B,
I-V plot from the same neuron as in
A shows a marked increase in
GN during hypoxia. C, Plot of
Idif from the data shown in
B.
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Figure 3.
Bath application of BaCl2 does not
affect the hypoxic response. A1,
B1, Traces recorded before, during, and 4 min after
hypoxia in the absence (A1) and presence
(B1) of BaCl2 (1 mM).
Note the substantial decrease in instantaneous current during perfusion
with BaCl2, whereas the hypoxia-induced increase in
GN and the block of inward relaxations
remains in the presence of BaCl2.
A2, B2,
I-V plots recorded before
(A2) and during (B2)
BaCl2 application (data from same neuron as in
A1 and B1,
respectively).
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In contrast to what is usually observed in hippocampal cells (Krnjevic
and Leblond, 1989; Leblond and Krnjevic, 1989), in TC neurons hypoxia
did not produce any significant effect on voltage-activated whole-cell
outward currents (IOUT)
(IOUT = 27 ± 49 pA; from the control
IOUT = 1482 ± 165 pA, measured at 30 mV;
n = 29) (Fig. 1A1,B1).
Intracellular Cl or Cs+
does not affect the hypoxia-induced changes in
GN, IBH, and
inward relaxations
In six neurons clamped at 65 mV, Cl was
used as the main anion in the electrode solution (KCl, 136 mM), and the effect of hypoxia on GN
(GN = 104 ± 41%),
IBH (IBH = 57 ± 7 pA), and the inward relaxations (71 ± 11%) was not
different from the one observed when recording with
KMeSO4-filled electrodes (Table 1). In these six cells the
reversal potential of the hypoxia-induced current(s) was 53.9 ± 6.4 mV.
In 20 neurons recorded with electrodes containing cesium gluconate
(n = 10) or CsF (n = 10), hypoxia still
produced a marked increase in GN
(GN = 91 ± 22%; p < 0.001), an inward shift in IBH
(IBH = 57 ± 20 pA; p < 0.05 from IBH = 95 ± 42 pA), and a
block of the inward relaxations (84 ± 10%) (Fig.
2A,B, Table 1). However, the reversal potential of
the hypoxia-evoked current(s) recorded with
Cs+-filled electrodes was shifted to a more
depolarized potential (38.6 ± 4.7 mV), a value similar to the
reversal potential of Ih in TC neurons
(McCormick and Pape, 1990a,b; Soltesz et al., 1991; Pape, 1996).
As is clearly shown in Figure 2B, the high-threshold
Ca2+ currents of TC neurons were not depressed
during a 3- to 4-min-long hypoxic challenge
(ICa = 5 ± 22%, measured at 10 mV;
n = 13). This is in contrast to hippocampal neurons
where high threshold Ca2+ currents are depressed in
the first 3 min of oxygen deprivation after a transient initial
potentiation (Krnjevic and Leblond, 1989).
Effect of Ba2+
To eliminate a possible contribution by the fast inward rectifier
present in TC neurons (Williams et al., 1997), we tested the effect of
BaCl2 on the hypoxic response of TC neurons. Bath application of BaCl2 (0.1-2 mM) failed to
produce any significant effect on the hypoxia-induced changes in
GN and IBH
(GN = 128 ± 14%,
IBH = 36 ± 9 pA in
BaCl2, compared with GN = 123 ± 11%, IBH = 33 ± 10 pA in
control conditions in the same eight neurons) (Fig. 3, Table
2). In two of these cells after 20 min
perfusion with BaCl2 containing saline, the concomitant
application of ZD 7288 (100 µM) abolished the hypoxic
response (data not shown).
Voltage-dependence of the Idif
In eight neurons clamped at 40 mV and recorded with
Cs+-filled electrodes in the presence of external
BaCl2 (1-2 mM), TEA (10 mM), and
4-AP (0.1 mM), we looked at the reversible effect of
hypoxia on the amplitude of the inward relaxations evoked by hyperpolarizing voltage steps (Fig.
4A) and compared it
with Idif (i.e., the difference in the
instantaneous current measured in control and during hypoxia).
Idif had a threshold for activation of
approximately 45 mV, reached a maximum at 95 mV, and had a
V1/2 of 77.6 ± 2.3 mV and a k
of 8.7 ± 0.9 (n = 8) (Fig. 4B, closed circles). In agreement with previous studies
(McCormick and Pape, 1990a,b; Pape, 1996; Soltesz et al., 1991), the
corresponding values for Ih were
V1/2 = 88.4 ± 2.1 and k = 8.8 ± 0.8 (n = 8) (Fig. 4B,
open circles). There was, therefore, a striking similarity between the k of Idif and that of
Ih (Fig. 4B), whereas the
V1/2 of Idif was 10 mV
more depolarized than that of Ih.
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Figure 4.
Effects of hypoxia on inward relaxations
(Ih) and voltage-dependence of
Ih and Idif.
A, Normalized amplitude of the inward relaxations
(Ih) shows the reversible depression
produced by hypoxia in eight neurons recorded with
Cs+-filled electrodes in the presence of
extracellular BaCl2 (1-2 mM), TEA (10-20
mM), and 4-AP (0.1 mM). Error bars represent
SEM. The three data sets were normalized using
Imax of the control data. B,
Normalized activation curve of Ih measured
in normoxic conditions ( ) and normalized amplitude of
Idif ( ) constructed from the same eight
neurons as in A. Error bars and curves represent SEM and
the Boltzmann curves, respectively (for details, see Materials and
Methods). Dashed lines point to the
V1/2 of the two curves. Note the similarity
in slope and the 10 mV difference in V1/2
between the two curves.
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Block of Ih depresses the
hypoxic response
Because of the similarity in the voltage-dependence and reversal
potential of Ih and
Idif, we tested the effects of ZD 7288, a
selective blocker of Ih (Harris and Constanti,
1995; Williams et al., 1997) in 11 TC neurons, where in control
conditions hypoxia had produced the usual increase in
GN (GN = 109 ± 18%) and inward shift in IBH
(IBH = 26 ± 6 pA). Bath application
of ZD 7288 (100-300 µM) blocked
Ih and significantly depressed the hypoxic changes in GN and IBH (by
88 ± 6 and 92 ± 9%, respectively) (Fig. 5, Table 2). Because the action of ZD
7288 on Ih is irreversible (Harris and
Constanti, 1995; Williams et al., 1997), no attempt was made to wash
out the effect of ZD 7288 on the hypoxic response.
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Figure 5.
ZD 7288 blocks the hypoxic response of TC neurons.
A, Examples of currents recorded in control, during, and
3 min after hypoxia in the absence (top row) and
presence (bottom row) of ZD 7288 (300 µM).
B, I-V plots (from the
same neuron as in A) obtained in the absence
(B1) and presence (B2) of
ZD 7288. C, Idif measured
before () and during ( ) bath application of ZD 7288 (from the
data shown in B1 and B2,
respectively).
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As a further test, five neurons were perfused with a solution
containing 2-3 mM CsCl, a reversible blocker of
Ih (McCormick and Pape, 1990a; Soltesz et al.,
1991). In these conditions, Ih was blocked but
hypoxia failed to produce an increase in GN
(GN = 16 ± 16%; n = 5), in contrast to the consistent GN increase seen in the same neurons during hypoxic tests in the absence of extracellular CsCl (GN = 113 ± 19%;
p < 0.01) (Fig. 6, Table 2). In two of these neurons that could be reliably clamped for a
sufficient period of time, the effect of hypoxia recovered after 15 min
washout of CsCl (GN = 67 and 82%) (Fig.
6).
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Figure 6.
Bath application of CsCl reversibly abolishes the
hypoxic response. A1, Examples of currents
evoked by hyperpolarizing voltage steps in the absence (top
row) and presence (bottom row) of CsCl (3 mM). A2, Traces from another TC
neuron recorded in the presence (top row) and after 15 min washout (bottom row) of CsCl (3 mM).
B1, B2,
I-V plots measured in the absence
(B1) and presence (B2) of
CsCl from the same neuron as in A.
B3, Idif recorded in
the absence () and presence () of CsCl (from the data shown in
B1 and B2, respectively).
C1, C2,
I-V plots in the presence
(C1) and after 15 min washout
(C2) of CsCl (3 mM) from the same
neuron as in A2. C3,
Idif measured in the presence () and
after 15 min washout of CsCl () (from the data shown in
C1 and C2,
respectively).
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It is well known that Ih is carried by
Na+ and K+ ions (McCormick and
Pape, 1990a; Soltesz et al., 1991). In our experiments, we reduced the
[Na+]o to 16 mM by
replacing NaCl with N-methyl-D-glucamine (134 mM). Under these conditions, Ih was
blocked and the hypoxia-induced changes in GN
and IBH were abolished
(GN = 10 ± 10%,
IBH = 14 ± 19 pA; p > 0.05 for both; n = 5), compared with a
GN = 99 ± 16%
(p < 0.01) and a IBH = 26 ± 8 pA (p < 0.05) in control
conditions. In the one neuron that could be clamped for a period
sufficiently long to allow the re-establishment of the control
[Na+]o, the hypoxic response
resumed (GN = 73%) (data not shown).
Sensitivity of the hypoxic response to
[Ca2+]o
Because metabolic arrest soon leads to a rise in cytosolic
Ca2+ (Hansen, 1985; Kaplin et al., 1996), it has
been suggested that Ca2+ could trigger the hypoxic
changes in membrane properties (Krnjevic, 1993; Belousov et al.,
1995).
In our experiments we tested the contribution of
Ca2+-mediated changes to the hypoxic response of TC
neurons by decreasing the [Ca2+]o to
0.5 mM and increasing the
[Mg2+]o to 8-10 mM and
concomitant bath application of the voltage-activated Ca2+-channel blockers NiCl2 (1 mM) and CdCl2 (300 µM). In
another five neurons the [Mg2+]o was
increased to 8-10 mM, whereas the
[Ca2+]o was left unchanged (2 mM). In all of these cells (n = 20), hypoxia failed to produce any significant changes in
GN and IBH (GN = 17 ± 13% and
IBH = 6 ± 8 pA) (Fig.
7, Table 2). In three of these neurons,
the hypoxic response recovered 15-20 min after returning to the
control solution (GN = 90 ± 27%,
IBH = 26 ± 5 pA) (Fig. 7).
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Figure 7.
Ca2+ dependence of the hypoxic
response. A, Camera lucida reconstruction of the neuron
(from which all data in this figure were obtained) shows the
characteristic morphology of a dLGN TC neuron. Whole-cell recording
with a biocitin-containing patch electrode obtained in standard ACSF
(B1, C1), then in solution
containing low Ca2+ (0.5 mM), high
Mg2+ (8 mM), NiCl2 (1 mM), and CdCl2 (300 µM)
(B2, C2), and during washout
with standard ACSF (B3, C3).
In all cases, examples of currents recorded before, during, and 4 min
after hypoxia are shown. C1,
C2, C3,
I-V plots obtained from the data shown
in B1, B2, and
B3. Note the marked depression of the hypoxic
response in low Ca2+/high Mg2+
solution. Inset graph in C1 shows
Idif measured before (), during (),
and after ( ) perfusion with the solution containing low
Ca2+ (from the data shown in
C1, C2, and
C3, respectively).
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Internally applied Ca2+ chelators
An increase of the EGTA concentration (to 10 mM) in
the internal solution had no effect on the hypoxia-induced changes in GN and IBH because 21 hypoxic tests in 11 neurons produced a 97 ± 29% increase in
GN, associated with an inward shift of
35 ± 17 pA in IBH and a 89 ± 10%
reduction of the amplitude of inward relaxations (Fig.
8, Table 1). Because of the fast and
pH-independent Ca2+-chelating ability, we also
tested BAPTA in 13 cells. BAPTA (10 mM in five cells, 30 mM in eight cells) failed to prevent the hypoxia-induced
increase in GN (GN = 99 ± 16%; n = 13), the inward shift in
IBH (IBH = 70 ± 20 pA; n = 13), and the depression of inward
relaxations (81 ± 14%; n = 13) (Fig. 8, Table
1). On the other hand, neurons recorded with BAPTA, but not with 10 mM EGTA, showed a significantly greater
GN in control conditions compared with neurons
recorded with the standard 1 mM EGTA in the intracellular
solution (BAPTA, GN = 8.7 ± 0.6 nS,
n = 13, p < 0.01 compared with 1 mM EGTA; 10 mM EGTA, GN = 3.8 ± 0.5 nS, n = 11) (Table 1).
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Figure 8.
Ca2+ chelators fail to produce
any significant effect on the hypoxic response.
A1, A2, Pen recorder traces
from two TC neurons recorded with electrodes containing EGTA (10 mM) (A1) or BAPTA (30 mM) (A2). The pen recorder speed was
accelerated (time calibrations in brackets in
A2) during the hyperpolarizing voltage steps
(from 65 to 85 mV) to visualize the changes in instantaneous and
steady-state current elicited during hypoxia.
B1, B2,
I-V plots obtained from the two other TC
neurons show the effect of hypoxia recorded with electrodes containing
EGTA (10 mM) and BAPTA, respectively.
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Inhibition of transmitter release
In four slices, the Ca2+-dependent release of
transmitters was blocked by preincubation (2-5 hr) with botulinum
toxin A (100 nM) (Sanchez-Prieto et al., 1987). In four
neurons, one in each of the four slices, hypoxia failed to produce any
significant effect on GN
(GN = 3 ± 15%),
IBH (IBH = 10 ± 6 pA), or the amplitude of the inward relaxations (12 ± 8%
increase) (Fig. 9B, Table 2).
Note that preincubation of the slices had no effect on the amplitude of
the inward relaxations (332 ± 53 pA; n = 4) (Fig.
9).
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Figure 9.
Lack of hypoxic response in TC neurons
preincubated with botulinum toxin A. A, Whole-cell
recording with an electrode containing KMeSO4 shows the
absence of any effect of hypoxia in a TC neuron from a slice
preincubated with botulinum toxin A (100 nM) for 4 hr.
B, I-V plot from the same
neuron as in A.
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Block of transmitter receptors
Because the sensitivity to botulinum toxin of the hypoxic response
of TC neurons suggested an involvement of presynaptic mechanisms, we
further assessed this hypothesis by testing the effect of antagonists for transmitter receptors whose activation is known to affect Ih in TC neurons. Combined application of
propanol (30 µM), cimetidine (50 µM), and
methysergide (30 µM) produced a significant reduction in
the effect of hypoxia on GN (58 ± 17%),
IBH (48 ± 2%), and Ih (45 ± 9%) in three TC neurons (each in
a different slice).
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DISCUSSION |
The main findings of this study in TC neurons of the rat dLGN are
that (1) brief periods of hypoxia cause an inward shift in
IBH, accompanied by an increase in
GN and a decrease in the amplitude of the inward
relaxations elicited by hyperpolarizing voltage steps, and (2) these
hypoxia-induced changes are Ca2+-dependent and
abolished by selective blockers of Ih and by
botulinum toxin but are unaffected by high concentrations of internally applied Ca2+ chelators. The simplest explanation of
these results is that hypoxia causes a 10 mV positive shift in the
voltage-dependence of Ih together with a change
in its kinetics that transforms Ih into a fast
activating current. An increase in Ca2+-dependent
release of transmitters may underlie some of these effects.
Mechanism of the hypoxia-induced changes in
GN and IBH
K+ and Cl channels
An increase in GN caused by the activation
of K+ currents during hypoxia by either a depletion
of intracellular ATP (Ben-Ari, 1990b; Fujimura et al., 1997) or an
increase in [Ca2+]i (Belousov et al.,
1995) has been shown in hippocampal slices. In our experiments,
however, no significant change in either voltage-activated outward
currents (in the range from 40 to 30 mV) or
IBH (at potentials more than or equal to 40
mV) was observed during hypoxia. In addition, internally applied
Cs+, which has been widely used as a powerful tool
for eliminating various K+ currents (Gay and
Stanfield, 1977; Cook, 1988; Halliwell, 1990), together with bath
applications of TEA, BaCl2, and 4-AP, failed to
affect the hypoxia-induced changes in GN,
IBH, and inward relaxations. The lack of
effect of BaCl2 also indicates that the hypoxia-induced depression of the inward relaxations was not secondary to a
K+ current activation as shown in substantia nigra,
zona compacta neurons (Watts et al., 1996) and eliminates a possible
contribution of a leak conductance (Hagiwara et al., 1978; Cook, 1988;
Halliwell, 1990) and the fast inward rectifier (Williams et al., 1997)
to the hypoxic response of TC neurons.
The reversal potential of the hypoxia-induced current, however, was
shifted from 57 to 37 mV when recording with
Cs+-filled electrodes, suggesting some contribution
by current(s) with a reversal potential less than 57 mV. In contrast
to previous results in the hippocampus (Zhang and Krnjevic, 1993;
Belousov et al., 1995), the lack of any difference in the effect of
hypoxia measured with either MeSO4-,
Cl-, F-, or gluconate-filled
electrodes excluded any significant contribution of
Cl currents to the hypoxic response. Together
these results indicates that current or currents with a reversal
potential less than 57 mV, possibly carried by K+
and blocked by internal Cs+, contribute to the
hypoxic response of TC neurons. No further attempt was made in this
study to identify this current(s).
Ih channels
A consistent block of the hypoxic response was observed with three
different manipulations that are known to abolish
Ih in TC neurons: bath applications of ZD 7288 (Williams et al., 1997) and of CsCl (McCormick and Pape, 1990a; Soltesz
et al., 1991) and a decrease in [Na+]o
(McCormick and Pape, 1990a). The reversibility of the block produced by
the last two procedures suggests that this abolishment of the hypoxic
response is attributable to a selective block of Ih and not to a rundown of the hypoxic response,
as has been observed in the hippocampus (Zhang and Krnjevic, 1993).
Although extracellular Cs+ has been shown to depress
the M-current (Coggan et al., 1994), which may also have been enhanced
by a hypoxia-induced rise in [Ca2+]i
(Yu et al., 1994), there is no study that indicates a similar effect of
ZD 7288 and low [Na+]o on this
voltage-activated K+ current.
Thus, the pharmacological sensitivity and the reversal potential
(measured with Cs+-filled electrodes) of
Idif are identical to those of
Ih, suggesting that the former current
may represent Ih that has undergone a 10 mV
positive shift in its voltage-dependence caused by hypoxia. This
interpretation accounts for the hypoxia-induced inward change in
IBH (observed at more depolarized potentials
than the normal activation range of Ih)
and for the increase in steady-state current. During hypoxia,
therefore, Ih might be activated and not
blocked, as the depression of the inward relaxations might indicate. In line with this suggestion, our explanation for the substantial increase
in instantaneous current and the resulting depression of the inward
relaxations is that hypoxia somehow changes the kinetics of
Ih, transforming it into a fast
activating current. Under these conditions, Ih
channels would open within a few milliseconds of the start of
hyperpolarizing voltage steps, explaining the large increase in
instantaneous current (as well as in GN)
and the depression of inward relaxations. Clearly, this scenario
represents the most parsimonious explanation of the present results,
but we cannot exclude the alternative possibility that in TC neurons hypoxia blocks Ih and concomitantly activates a
novel, very fast activating current that has a voltage-dependence
similar to, and pharmacological properties and an ionic permeability
identical to, those of Ih.
What activates Ih during hypoxia?
Ca2+-dependence of the hypoxic response
The hypoxic response of TC neurons was found to be highly
sensitive to [Ca2+]o, although
there is no direct evidence from TC neurons, measurements of
Ca2+ influx or
[Ca2+]i in other brain regions have
shown that hypoxia can elicit an increase in cytosolic
Ca2+ (Hansen, 1985; Kass and Lipton, 1986; Dubinsky
and Rothman, 1991; Kaplin et al., 1996). This increased
[Ca2+]i may play a role both
presynaptically by increasing transmitter release and postsynaptically
by directly activating Ih (Hagiwara and
Irisawa, 1989; Ingram and Williams, 1996) or the other current(s) responsible for the hypoxia-mediated effects in TC neurons.
In our experiments, we did not observe any significant effect of
hypoxia on high voltage-activated Ca2+ currents. In
addition, the lack of action of high concentrations of internally
applied Ca2+ chelators does not support a
postsynaptic origin of the Ca2+-dependence of the
hypoxic response, although the effectiveness of these chelators would
be somewhat limited if the hypoxia-activated channels were locally
regulated by Ca2+ released from internal stores
close to the surface membrane or if Ca2+ would enter
the neuron via channels located in close proximity to the
hypoxia-activated channels. The increase in resting
GN observed when using BAPTA (cf. Zhang et al.,
1995) may indicate that a K+ conductance, which is
normally suppressed by the normal
[Ca2+]i, is activated when
BAPTA lowers [Ca2+]i below a critical
level, as is the case for r-eag type of gK (Stansfeld et al., 1996).
The lack of action of internally applied Ca2+
chelators and the block by botulinum toxin, an agent known to inhibit
Ca2+-dependent transmitter release (Sanchez-Prieto
et al., 1987), suggest that the Ca2+-dependence of
the hypoxic response of TC neurons is likely to have a presynaptic
origin, i.e., during hypoxia the Ca2+-dependent
release of transmitters may increase, therefore leading indirectly to
some of the observed effects of hypoxia. In ischemic conditions, (i.e.,
lack of oxygen and glucose) Ca2+-independent release
of transmitters is increased because of reverse operation of the uptake
system, secondary to a reduced Na+ electrochemical
gradient (Kauppinen et al., 1988; Nicholls and Attwell, 1990;
Szatkowski and Attwell, 1994). Depletion of the [ATP]i, which is essential for the
Na+/K+ exchanger, is the major
factor responsible for a reduced Na+ electrochemical
gradient. On the other hand, oxygen deprivation alone, i.e., not
accompanied by hypoglycemia, causes only very minor changes in
[ATP]i (Madl and Burgesser, 1993), indicating that the
Ca2+-dependent transmitter release may be
upregulated during brief periods of hypoxia.
Might transmitters activate Ih
during hypoxia?
The results of the above experiments indicate that an increase in
transmitter release might underlie some of the effects of hypoxia in TC
neurons. Interestingly, several neurotransmitters, including
noradrenaline, serotonin (McCormick and Pape, 1990b; Soltesz et al.,
1991), histamine (McCormick and Williamson, 1991), and nitric oxide
(Pape and Mager, 1992; for review, see Pape, 1996) are known to
increase Ih in TC neurons by eliciting a
positive shift in its steady-state activation curve. This action gives rise to an inward current at potentials around 60 mV and an increase in steady-state current elicited by hyperpolarizing voltage steps (McCormick and Pape, 1990b; Soltesz et al., 1991; Pape, 1996). The size
of this inward current and the magnitude of this increase in
steady-state current are comparable to the shift in
IBH and to the increase in steady-state current,
respectively, evoked during hypoxia. It is reasonable, therefore, to
suggest that a hypoxia-mediated release of one or more of these
transmitters may be responsible for some of the effects elicited by
hypoxia in TC neurons. Indeed, strong support for this hypothesis is
provided by the finding that a combined block of noradrenaline ( ),
serotonin, and histamine (H2) receptors produced
a substantial reduction of the hypoxic response.
Although an increase in the activation rate of
Ih in the presence of these transmitters has
been reported (McCormick and Pape, 1990a,b; McCormick and Williamson,
1991), none of them, applied alone, has been shown to be
able to produce as large a change in the kinetics properties of
Ih as to transform it into a very fast
activating (i.e., instantaneous) current during hypoxia. Interestingly,
each of these transmitters increases the instantaneous current slightly
(compare Fig. 5, A and C, in McCormick and Pape, 1990b; and Fig. 6, A and B, in McCormick and
Williamson, 1991), and forskolin, which mimics the effects of
noradrenaline and serotonin on Ih in TC neurons
(Pape, 1996), appears to be able to transform IQ
of sympathetic neurons into an instantaneous current (D. A. Brown,
personal communication). Nevertheless, several possibilities for the
large, hypoxia-mediated change in Ih kinetics
remain to be elucidated: (1) a synergistic action of known thalamic
transmitters, possibly involving both cAMP and cGMP; (2) the action of
some unknown transmitter(s) released during hypoxia; and (3) the
additional action of hypoxia-mediated postsynaptic effects, such as
changes in intracellular pH or cell swelling.
Pathophysiological role of Ih
activation during hypoxia
It has been shown that hypoxia/ischemia causes highly organized,
system-preferential, topographic encephalopathy and targets regions
that play a pivotal role in sensory integration. Injury mediated by
oxygen deprivation is found preferentially in the somatosensory cortex,
the basal ganglia (including putamen and subthalamic nucleus), the
ventral thalamus, the medial and dorsal LGN, and the tectal nuclei
(Martin et al., 1997). The hypoxia-mediated inward current and increase
in conductance observed in this study will affect the amplitude and
kinetics of synaptic potentials generated in, and as a consequence the
output of, TC neurons. These effects may be carried forward from the
thalamus to the cortex via corticothalamic connections (Jones, 1985;
Salt et al., 1995;) and then to the striatum and the subthalamus via
corticosubthalamic projections (Fujimoto and Kita, 1993; Bevan et al.,
1995; Jones et al., 1977), explaining the topographic cascade of
transneuronal injury in brain areas involved in sensorimotor
integration.
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FOOTNOTES |
Received Jan. 13, 1998; revised April 28, 1998; accepted May 1, 1998.
This work was supported by the Wellcome Trust (Grant 37089). We thank
Bob Jones for photography and Tim Gould for technical assistance with
some experiments.
Correspondence should be addressed to V. Crunelli, Physiology Unit,
School of Molecular and Medical Biosciences, University of Wales
Cardiff, Museum Avenue, Cardiff, CF1 3US, UK.
| |
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Top
Abstract
Introduction
