 |
INTRODUCTION |
It has already been reported that rat midbrain dopaminergic "principal" neurons hyperpolarize during a brief hypoxic stimulus (Mercuri et al. 1994a
,b
) through an activation of K+ conductances. This hypoxic response is similar to that observed in other central neurons (Ben-Ari 1992
; Grigg and Anderson 1989
; Jiang et al. 1992
; Luhmann and Heinemann 1992
; Mourre et al. 1989
; Nieber et al. 1995
; Stanford and Lacey 1996
; Trapp and Ballanyi 1995; Wu et al. 1996
) where it is mainly attributed to an activation of potassium ATP-inhibitable (K+ATP) channels. In spite of the fact that the substantia nigra (SN) has the highest density of glibenclamide binding sites (Bernardi et al. 1988
; Hicks et al. 1994
; Mourre et al. 1989
; Xia and Haddad 1991
), which indicate the presence of K+ATP channels, and that electrophysiological studies have already shown the presence of sulphonylurea-sensitive K+ATP channels on the DAergic cells (Hausser et al. 1991
; Röper and Ashcroft 1995
; Seutin et al. 1996
; Stanford and Lacey 1995
; Watts et al. 1995
), the cellular response of the principal neurons to O2 deprivation does not seem to be exclusively mediated by the activation of sulphonylurea-sensitive K+ channels (Mercuri et al. 1994a
,b
). Our previous electrophysiological data on the effects of hypoxia on the DAergic cells (Mercuri et al. 1994a
,b
) are not fully consistent with patch-clamp reports demonstrating sulphonylurea-sensitive K+ channels, activated by hypoxia in these neurons (Jiang et al. 1994
).
Considering the apparent discrepancies between our observations and the results of other groups we performed both sharp microelectrode intracellular and whole cell patch-clamp recordings on SN compacta (SNc) and ventral tegmental area (VTA) DAergic neurons, to re-examine the effects of hypoxia. In particular, we tested whether or not the hyperpolarization/outward response induced by oxygen deprivation might also be modulated by sulphonylureas. We also compared the biophysical properties of the hypoOUT and posthypoOUT to determine if either the dialysis of the cytosol or the modification of the intracellular content of ATP could modify the pattern of the membrane responses associated with a metabolic failure in the DAergic cells.
 |
METHODS |
Preparation of the tissue
The method used has been described previously (Mercuri et al. 1994a
,b
, 1995
). In brief, horizontal slices comprehending the substantia nigra and the ventral tegmental area were cut from the ventral mesencephalon of Wistar rats (100-250 g), anesthetized with halothane, and killed. The brain was removed and horizontal slices (thickness 200-300 µm) were cut by a vibratome starting from the ventral surface of the midbrain. A single slice was then transferred into a recording chamber and completely submerged in an artificial cerebrospinal fluid with continuously flowing (2.5 ml/min) solution at 35-36°C, pH 7.4. This solution contained (in mM) 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 18 NaHCO3, gassed with 95% O2-5% CO2 (pH 7.4).
Intracellular recordings
Intracellular recording electrodes (Clark 1- to 1.5-mm-thick wall), pulled by Narishige vertical and horizontal pullers, were filled with 2 M KCl and had a tip resistance of 20-40 M
. The signals were obtained by an amplifier (Axoclamp-2A, Axon Instruments) in a bridge or single-electrode voltage-clamp mode. Under single-electrode voltage-clamp, the switching frequency was 3-4 Hz and a duty cycle of 30% was used. The headstage voltage was continuously monitored on a separate oscilloscope to ensure sufficient decay of the electrode transient.
Patch-clamp recordings
One slice was transferred to a recording chamber on the stage of an upright microscope (Axioscope, Zeiss). The dopaminergic neurons were visualized by infrared video imaging (Hamamatsu, Japan) and approached by applying positive pressure. Whole cell recordings were made using pipettes fabricated from borosilicate glass (WPI 1.5 mm) and pulled with a PP 88 Narishige puller. The pipette solution contained (in mM) 128 K+-gluconate, 10 KCl, 0.3 CaCl2, 1 MgCl2, 10 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (HEPES), 1 ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
(EGTA), 2 Mg-ATP, and 0.25 Na3-guanosine 5
-triphosphate (GTP) (pH 7.3) and had resistance of ~4 M
. Whole cell recordings were performed from DAergic neurons as described by Bonci and Williams 1996
, with an Axopatch 1-D amplifier (Axon Instruments).
Hyperpolarizing and depolarizing voltage commands (see figure legends for details) lasting 200-400 ms from a holding potential of
60 mV were delivered to induce series of currents, which were used to construct the I-V curves. The membrane voltage and current were recorded and stored on computers with the pClamp software 6 (Axon Instruments) and with the MacLab analog/digital interface and Chart software (AD Instruments). The data were subsequently analyzed with an Axograph (Axon Instruments) and an Origin (Microcal) program.
Hypoxia was transiently caused by the superfusion of a solution equilibrated with 95 % N2-5% CO2. Drugs were bath applied at known concentrations via a three-way tap system. A complete exchange of the solution in the recording chamber occurred in ~1 min.
The following drugs were used: dopamine hydrochloride, tolbutamide (Sigma), glibenclamide and glipizide (Research Biomedical International), and charybdotoxin (Alomone Labs). Stock solutions of the sulphonylureas were made in dimethyl sulfoxide and diluted to the appropriate concentrations. The vehicle had no effect when applied alone.
The numerical data were expressed as means ± SE of the mean. The statistical significance of the data (P < 0.05) was evaluated by Student's t-test.
 |
RESULTS |
The principal dopaminergic cells of the ventral mesencephalon, under bridge and whole cell current-clamp mode, were identified by their spontaneous firing (1-6 Hz), a hyperpolarizing/outward response to dopamine application (10-30 µM) (not shown) and a hyperpolarization-activated inward rectification of the membrane (Ih) under voltage-clamp mode (Figs. 1B, 3B, and 4B). The characteristics of the recorded neurons met the criteria of previously described "principal" DAergic cells (Lacey et al. 1989
; Mercuri et al. 1995
), which were stained for cathecolamines (Grace and Onn 1989
; Jiang et al. 1994
; Yung et al. 1991
).

View larger version (25K):
[in this window]
[in a new window]
| FIG. 1.
Hypoxic responses of intracellularly recorded dopaminergic neurons. A: 2 different periods of hypoxia, indicated by bars, induced outward currents which were followed by a posthypoxic response. Ba: currents induced by voltage steps ( 45 to 115 mV, 10 mV increments), from a holding potential of 60 mV. Traces were obtained at time points (1-4) reported in A. Note the increase in membrane conductance during hypoxic and posthypoxic periods (2 and 3). Bb: mean current-voltage relationship for dopaminergic cells (n = 4) in control condition and during hypoxic and post-hypoxic currents. Current amplitudes to construct I-V plots were taken at offset of capacitance transients and before development of Ih current.
|
|

View larger version (24K):
[in this window]
[in a new window]
| FIG. 3.
Responses to hypoxia of dopaminergic cells patch-clamped in whole cell configuration. Pipette solution contained 2 mM ATP. Outward current caused by hypoxia in a dopaminergic cell. B: currents elicited by depolarizing and hyperpolarizing voltage steps ( 45 to 135 mV, 15-mV increment, holding potential 60 mV), at time points indicated in A before (1), during (2), and after (3) hypoxia. Note that current pulses were blanked in A. C: current-voltage relationship obtained before ( ) and during hypoxia ( , n= 6). Difference between 2 curves ( ) represents hypoxia-induced current under whole cell recordings.
|
|

View larger version (21K):
[in this window]
[in a new window]
| FIG. 4.
Tolbutamide abolishes hypoxic outward current in patch-clamped neurons (pipette solution containing 2 mM ATP). A: reversible blockade of hypoxic current caused by tolbutamide (100 µM). Numbers (1-4) on top of traces represent time points at which currents in B were elicited; current transients were blanked. B: currents evoked by depolarizing and hyperpolarizing voltage steps ( 45 to 135 mV, 15-mV increment, holding potential 60 mV). Note that conductance increase during hypoxia (2) was also reversibly depressed (3) by tolbutamide (100 µM). Broken lines indicate zero current. C: bars indicate mean hypoxia-induced current in patch-clamped neurons, in control condition and in presence of tolbutamide (100 µM). Mean amplitudes were 58.3 ± 13.2 pA and 5 ± 11.7 pA, respectively (n = 8).
|
|
Effects of hypoxia on intracellularly recorded neurons
Figure 1A illustrates typical current responses to hypoxia of a DAergic neuron held at
60 mV, under conventional single-electrode voltage-clamp conditions. The brief hypoxia elicited a complex outward current consisting of at least two phases. The first one started within 1.5-2 min and reached a plateau in 2-3 min. We referred to this current as "hypoxic outward" (hypoOUT). The second one appeared upon return to normoxic Ringer. This posthypoxic outward current (posthypoOUT) developed in most cells, reached a peak within 0.5-1.5 min and recovered within 3-5 min. This current had an absolute amplitude (measured from the prehypoxic baseline) higher than that of the hypoOUT. The mean amplitude of the hypoOUT was 110.2 ± 15.2 pA (n = 18), whereas the mean amplitude of the posthypoOUT was 149.6 ± 10.6 pA (n = 18). Current traces evoked by stepping the voltage from a holding potential of
60 mV to command potentials in the range from
45 to
115 mV, showed a reversible increase in membrane conductance during the development of hypoOUT, which also persisted during the posthypoOUT (Fig. 1Ba) (Mercuri et al. 1994b
). Figure 1Bb illustrates the I-V relationship of the currents obtained during control condition, the development of the hypoxic and the posthypoxic responses in four dopaminergic cells. Note that the slope of the hypoxic curve (
) increased as expected for an augmentation in slope conductance. The mean reversal potential for the hypoOUT was
83.7 ± 3.8 mV (n = 4). The posthypoxic curve (
) did not intersect with the control one and had a parallel outward shift when it was compared with the hypoxic I-V relationship.
Tolbutamide, glibenclamide, and other potassium-channel blockers reduce the hypoxic current
Tolbutamide (100 µM), a known blocker of the K+ATP channels (Edwards and Weston 1993
), reduced the hypoOUT by 47.6 ± 7.7% of control (n = 16) (P < 0.001) (Fig. 2, A and C). There was a notable variability in sensitivity between neurons, with the hypoOUT either almost unaffected or completely blocked by tolbutamide in different cells. The reversal potential of the current remaining after tolbutamide treatment was
78 ± 3.4 mV, n = 5. We also tested whether or not specific K+ATP channel blocker glibenclamide (30 µM) could affect the hypoxic responses. It was found that this compound reduced the hypoOUT by 54.18 ± 7.5%(n = 3) (Fig. 2B). The effects of glibenclamide did not reverse. As already reported (Mercuri et al. 1994b
), extracellular barium (1 mM), a nonspecific blocker of K+ channels, strongly reduced the hypoOUT by 75 ± 10.8 % (n = 6, P < 0.001) (Fig. 2C). In addition, the large Ca2+-dependent K+ (BKCa) channels blocker, charybdotoxin (30-50 nM), reversibly depressed the hypoOUT after tolbutamide treatment (n = 3) (Fig. 2D).

View larger version (13K):
[in this window]
[in a new window]
| FIG. 2.
Effects of tolbutamide, glibenclamide, barium and charybdotoxin on hypoxic current. A: single-electrode voltage-clamp recordings from a dopaminergic cell. Outward current elicited by hypoxia was depressed in a reversible manner by tolbutamide (100 µM) (holding potential 60 mV). B: irreversible reduction of hypoxia-induced current caused by glibenclamide (30 µM) in another cell voltage clamped by a sharp microelectrode at 59 mV. C, top: depressant effect of tolbutamide (100 µM) on amplitude of hypoxic current. C, bottom: depressant effect of extracellular barium (1 mM). Values represent means ± SE of 6 samples (P < 0.05). D: tolbutamide-resistant current caused by hypoxia was depressed by charybdotoxin (30 nM). Wash out was obtained after 60 min (holding potential 60 mV).
|
|
Tolbutamide does not affect the posthypoxic current
As indicated by the analysis of the current-voltage relationship (Figs. 1 and 2), the peak of the posthypoOUT represents the superposition of the recovering hypoOUT and a current that is not associated with a change in slope conductance. To eliminate the contribution of hypoOUT to the posthypoOUT, we calculated the difference between the peak value of the posthypoOUT (149.6 ± 10.6 pA) and that of the hypoOUT (110.2 ± 15.2 pA). Thus the mean amplitude of this isolated posthypoOUT was 39.6 ± 9.2 pA (n = 18). Neither BaCl2 (1 mM) nor tolbutamide (100 µM) affected the amplitude of this isolated component of posthypoOUT(P > 0.05).
Effects of hypoxia on whole cell patch-clamped neurons
As already demonstrated by the recordings obtained with sharp microelectrodes, hypoxia induced a hyperpolarization (9.6 ± 0.3 mV, n = 12) in whole cell recordings under current-clamp conditions, thus preventing the spontaneous firing. These effects recovered completely after 2 min of reoxygenation (Fig. 5A). Under whole cell voltage-clamp at
60 mV, hypoxia induced a slowly activating hypoOUT (Fig. 3A), that reached a plateau in ~4 min (70.2 ± 14.5 pA, n = 8). The hypoOUT recovered upon reoxygenation in 2-3 min. A small posthypoOUT (16.6 ± 5 pA) was detected in a few cells (n = 4 out of 14, not illustrated). Figure 3B shows the currents evoked by stepping from
45 to
135 mV (holding potential was
60 mV) before, during, and after hypoxia. Note the increase in membrane conductance and the shift above zero (···) of the holding current caused by hypoxia. Figure 3C shows an averaged I-V plot of six cells and the current caused by hypoxia along the voltage range. The reversal potential for the hypoOUT was
87.9 ± 5.1 mV (n = 8).

View larger version (26K):
[in this window]
[in a new window]
| FIG. 5.
Effects of barium and glibenclamide on response to hypoxia. A: current-clamp recording showing effects of extracellular barium (300 µM) on both hyperpolarization and inhibition of firing induced by hypoxia. Cell is recorded in whole cell configuration; pipette solution contained 2 mM ATP. B: voltage-clamp recording of membrane current in another dopaminergic cell. Note that both barium (300 µM) and glibenclamide (30 µM) depress hypoxic outward current (holding potential 60 mV).
|
|
Sulphonylureas block the hypoxia-induced outward current
Differently from the experiments done with sharp microelectrodes, under patch-clamp conditions, the hypoOUT and the associated increase in membrane conductance were almost completely blocked by sulphonylureas, such as tolbutamide (100 µM; 8 cells) (Fig. 4), glibenclamide (30 µM) (2 cells; Fig. 5B), and glipizide (100 nM; 4 cells, not shown). Figure 4A shows the response to hypoxia in control condition and when 100 µM tolbutamide was added to the superfusate. The hypoOUT was abolished by the drug and recovered after ~15 min wash out. The blocking effects of glibenclamide and glipizide did not reverse even after >60 min of wash.
The hyperpolarization/outward current, induced by hypoxia, were also prevented by 300 µM BaCl2 (Fig. 5). The experiment illustrated in Fig. 5B demonstrates that barium and glibenclamide efficiently depress hypoOUT even when it was already induced.
ATP sensitivity of the hypoxia-induced hyperpolarization/outward current
The concentration of intracellular ATP is thought to decrease during an hypoxic episode (Kass and Lipton 1989
; Lipton and Whittingham 1982
). The reduction of cytosolic ATP levels (Hausser et al. 1991
; Stanford and Lacey 1995
; Watts et al. 1995
) together with an increase in intracellular Ca2+ (Jiang et al. 1994
) might promote the opening of the K+ATP channels. To test for an involvement of cytosolic ATP, we performed patch-clamp recordings by dialyzing the intracellular environment with a solution in which the ATP concentration was increased from 2 to 10 mM.
The intracellular dialysis with 10 mM ATP reduced both the hypoOUT (Fig. 6) and the membrane hyperpolarization (not shown). In fact, the mean amplitude of the hypoOUT was 5.6 ± 5.1 pA (n = 9) in 10 mM ATP in comparison with 70.2 ± 14.5 pA (n = 8) in 2 mM ATP (Fig. 6B). The dopamine-induced hyperpolarization was, however, unaffected by the presence of high concentration of this nucleotide triphosphate (not shown). In fact, DA (30 µM) caused 9.8 ± 1.2 mV (n = 3) and 10.3 ± 0.3 mV (n = 3) hyperpolarization with 2 and 10 mM ATP in the pipette, respectively.

View larger version (12K):
[in this window]
[in a new window]
| FIG. 6.
Hypoxia-induced outward current is much smaller in neurons perfused with a solution containing 10 mM ATP. A: top: typical outward current in response to hypoxia of a patch-clamped neuron intracellularly perfused with a solution containing 2 mM ATP. Bottom: response to hypoxia recorded from another cell perfused with a 10 mM ATP-containing pipette solution. Note different current scale for 2 traces (holding potential 60 mV). B: hypoxia-induced currents in patch-clamped neurons intracellularly perfused either with 2 mM ATP or 10 mM ATP. Mean current amplitude values were 70.2 ± 14.5 pA (n = 8) and 5.6 ± 5.1 pA (n = 9), respectively.
|
|
 |
DISCUSSION |
We and others have already reported that the principal dopaminergic cells of the ventral mesencephalon are hyperpolarized and the generation of spontaneous action potentials is inhibited by hypoxia. This response is mainly due to the activation of K+ channels. However, although some authors have shown that an activation of K+ATP channels (Jiang et al. 1994
) is the main effect of hypoxia on the DAergic cells, we have previously determined (Mercuri et al. 1994b
) that the hypoxic response is not abolished in the presence of the K+ATP channels antagonist, glibenclamide. Taken together, all these data have some points that are worth re-examining.
Sulphonylureas-sensitive and -insensitive hypoxic current
The hypoOUT in intracellularly recorded neurons is reduced by the K+ATP channel blockers tolbutamide and glibenclamide. Thus it is clear that a significant component of the hypoOUT is generated by K+ channels sensitive to both compounds. Our previous underestimation of a sulphonylurea-sensitive component of the hypoOUT likely depended on a) the few experiments performed with the K+ATP channel blocker, glibenclamide, and b) the cell-to-cell variation in the amount of the sulphonylurea-sensitive outward current evoked by hypoxia. Accordingly, it has been reported that the amplitude of the tolbutamide-sensitive hyperpolarization caused by hypoxia varies between CA1 hippocampal and cortical neurons (Fujimura et al. 1997
; Luhmann and Heinemann 1992
). Notably, the blockade of part of the hypoOUT by tolbutamide does not affect its reversal potential. This implies that also the tolbutamide-resistant component of the hypoOUT is carried out by K+.
A peculiar observation is that tolbutamide, glibenclamide, and glipizide completely prevented the hypoOUT in patch-clamped principal neurons. In fact, the values of the tolbutamide-sensitive currents obtained with sharp microelectrodes (55.5 ± 8.5 pA) and the value of the hypoOUT obtained with patch electrodes (70.2 ± 14.5 pA) were quite similar (P > 0.05). This suggests that although certain components of the hypoOUT are lost during whole cell recording, this technique is well suited for an analysis of the sulphonylurea-sensitive outward current caused by hypoxia. Furthermore, the almost linear I-V relationship and the reversal potential of the sulphonylurea-sensitive hypoOUT are consistent with an increased activation of K+ channels during hypoxia, which can be blocked by sulphonylureas and extracellular barium. Indeed, it has recently been reported (Jiang et al. 1994
) that hypoxia prevalently opens a slightly rectifying 250 pS K+ATP channel on DAergic cells.
The experimental observation that raising the ATP concentrations in the pipette solution prevented the hypoOUT suggests that one of the factors that mediates the opening of K+ATP channels is a transient reduction of the intracellular level of this nucleotide. Although inside-out patches of dopaminergic cells have demonstrated that the sensitivity of K+ATP channels to internal ATP is high (Jiang et al. 1994
), the intracellular diffusion of (2 mM) ATP from the recording electrode could not be sufficient to prevent the hypoxia-induced activation of the K+ATP channels at distant sites. Thus an higher concentration of ATP (10 mM) in the micropipette should be used to completely load the cells. The prevention of the hypoOUT by supply of a high ATP concentration via the pipette also rules out the possibility that hypoOUT is mediated by sulphonylurea-sensitive but ATP-insensitive K+ channels (Seutin et al. 1996
). The finding, that hypoxia evokes a sulphonylurea-sensitive current, correlates well with anatomic studies that have clearly demonstrated binding sites for sulphonylureas on the soma and dendrites of dopaminergic neurons (Mourre et al. 1990
; Treherne and Ashford 1991
).
Tolbutamide-resistant hypoxic current
It would be interesting to speculate about the nature of the remaining hypoOUT after tolbutamide treatment in single-electrode voltage-clamp recordings. We have previously demonstrated that most of this current is blocked by extracellular barium, reduced by lowering the extracellular content of calcium and sodium, but it is insensitive to the blocker of the Ca2+-dependent small K+ channel (BKCa), apamin (Mercuri et al. 1994b
). In the present study we report that the sulphonylurea-resistant hypoOUT is reduced after the application of the large Ca2+-dependent K+ channel blocker, charybdotoxin (Latorre et al. 1989
). This supports the hypothesis that Ca2+-dependent K+ channels (Leblond and Krnjevic 1989
; Yamamoto et al. 1997
) of large conductance type generate the sulphonylurea-resistant hypoOUT.
Because the tolbutamide-insensitive component of the hypoOUT was only detected with sharp microelectrode, it is evident that a very intact cytoplasmic environment is essential for the full expression of the membrane response to the hypoxic stimulus. One argument in favor of this hypothesis is that the dialysis of intracellular milieu under whole cell recordings could wash out some cytosolic factors which are essential for the full expression of the hypoOUT. Interestingly, it has recently been reported that the amplitude of the hyperpolarizing response to hypoxia in CA1 hippocampal neurons is inversely related to the resistance of the recording electrode (Yamamoto et al. 1997
). The different experimental approaches used in studying the hypoxic response on dopaminergic neurons might account for the lack of detection of the sulphonylurea-resistant component when patch-clamp recordings are used.
Characterization of the posthypoxic current
Although analysis of the current-voltage relationship indicates an increase in conductance during the posthypoOUT, there is no further increase in conductance between hypoOUT and posthypoOUT. Thus the total amplitude of the posthypoOUT probably represents a superposition of the not yet recovered hypoxia-activated conductance and the reactivation of an electrogenic pump (Luhmann and Heinemann 1992
). In fact, the parallel shift of the posthypoxic curve with respect to the hypoxic one and the insensitivity to blockers of K+ channels suggests that an additional outward current during reoxygenation is very likely generated by the reactivation of the Na+-K+ pump (Fujiwara et al. 1987
; Mercuri et al. 1994a
).
The smaller and inconsistent expression of posthypoOUT observed in whole cell recordings could be due either to the buffering or to the disturbance of cytoplasmic factors.
Functional role of the hypoOUT
If the membrane hyperpolarization/outward current caused by hypoxia is a defensive response of the DAergic cells (Mercuri et al. 1994a
,b
), these neurons certainly possess more than one mechanism (sulphonylurea-sensitive and -insensitive) to reduce the deleterious consequences of an hypoxic episode.