Physiologisches Institut, Georg-August-Universität Göttingen, D-37073 Gottingen, Germany
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ABSTRACT |
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Haller, M., S. L. Mironov, and D. W. Richter. Intrinsic Optical Signals in Respiratory Brain Stem Regions of Mice: Neurotransmitters, Neuromodulators, and Metabolic Stress. J. Neurophysiol. 86: 412-421, 2001. In the rhythmic brain stem slice preparation, spontaneous respiratory activity is generated endogenously and can be recorded as output activity from hypoglossal XII rootlets. Here we combine these recordings with measurements of the intrinsic optical signal (IOS) of cells in the regions of the periambigual region and nucleus hypoglossus of the rhythmic slice preparation. The IOS, which reflects changes of infrared light transmittance and scattering, has been previously employed as an indirect sensor for activity-related changes in cell metabolism. The IOS is believed to be primarily caused by cell volume changes, but it has also been associated with other morphological changes such as dendritic beading during prolonged neuronal excitation or mitochondrial swelling. An increase of the extracellular K+ concentration from 3 to 9 mM, as well as superfusion with hypotonic solution induced a marked increase of the IOS, whereas a decrease in extracellular K+ or superfusion with hypertonic solution had the opposite effect. During tissue anoxia, elicited by superfusion of N2-gassed solution, the biphasic response of the respiratory activity was accompanied by a continuous rise in the IOS. On reoxygenation, the IOS returned to control levels. Cells located at the surface of the slice were observed to swell during periods of anoxia. The region of the nucleus hypoglossus exhibited faster and larger IOS changes than the periambigual region, which presumably reflects differences in sensitivities of these neurons to metabolic stress. To analyze the components of the hypoxic IOS response, we investigated the IOS after application of neurotransmitters known to be released in increasing amounts during hypoxia. Indeed, glutamate application induced an IOS increase, whereas adenosine slightly reduced the IOS. The IOS response to hypoxia was diminished after application of glutamate uptake blockers, indicating that glutamate contributes to the hypoxic IOS. Blockade of the Na+/K+-ATPase by ouabain did not provoke a hypoxia-like IOS change. The influences of KATP channels were analyzed, because they contribute significantly to the modulation of neuronal excitability during hypoxia. IOS responses obtained during manipulation of KATP channel activity could be explained only by implicating mitochondrial volume changes mediated by mitochondrial KATP channels. In conclusion, the hypoxic IOS response can be interpreted as a result of cell and mitochondrial swelling. Cell swelling can be attributed to hypoxic release of neurotransmitters and neuromodulators and to inhibition of Na+/K+-pump activity.
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INTRODUCTION |
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The intrinsic optical signal (IOS)
provides a noninvasive technique for imaging "neuronal functions"
in living brain. The signal originates from changes in the refractory
indexes of the cytoplasm and the extracellular space that together
determine light transmittance and/or scattering. As the signal
originates from endogenous processes in the tissue, it does not require
any cell labeling and is thus not subject to bleaching. The IOS imaging technique has been applied to the intact brain as well as to slice preparations. In the intact brain, IOS has been used, e.g., to map the
spatial distribution and patterning of neuronal activity (Holthoff and Witte 1998) as well as to verify
propagation of seizure (Federico et al. 1994
). In slices
from hippocampus, neocortex, and the retina, the IOS technique was used
to examine various pathological processes, such as spreading depression
in the retina (Fernandes de Lima et al. 1997
;
Ulmer et al. 1995
) and the hippocampus (Muller
and Somjen 1998
; Obeidat and Andrew 1998
) or
excitotoxicity originating from enhanced release of excitatory
neurotransmitters (Andrew et al. 1999
).
The mechanisms underlying the IOS are complex, and its origin is as yet
not completely understood. There are strong indications that an
essential component of IOS is determined by changes in cell volume
(Andrew et al. 1996; Polischuk and Andrew
1996
), which might originate from cell swelling due to
Na+, Ca2+,
Cl- accumulation and concomitant water fluxes
through the plasma membranes (Andrew and MacVicar 1994
)
or from swelling of adjacent glial cells buffering extracellular
K+ that is released during neuronal activity
(MacVicar and Hochman 1991
). However, there is also
evidence that not all components of the IOS can be explained in terms
of cell volume changes. Buchheim et al. (1999)
found
that electrical hyperactivity in hippocampal slices of rat leads to
diverse changes in the IOS: the IOS also increased during shrinkage of
the extracellular space, indicating the presence of a
volume-independent mechanism of IOS changes. Similarly, in hippocampal
slices (Aitken et al. 1999
), light transmittance was
found to increase during moderate hypotonia, but to decrease during
pronounced arterial hypotonia even though cells continued to swell.
Another puzzling information is that spreading depression (induced, for
example, by potassium injection) and hypoxic spreading depression-like
depolarization (induced by oxygen withdrawal) lead to a decrease in
transmitted light intensity (Muller and Somjen 1999
).
A possible explanation for all these divergent phenomena could be that
in addition to cell volume changes, IOS is also affected by changes in
the distribution and/or volume of subcellular structures. Objects such
as dendritic spines, dendritic beads and cellular organelles may change
their shape as water enters or leaves the cell. Specifically beading of
the dendritic arbor, i.e., the formation of varicosities in dendrites
as a result of an excitotoxic insult (Andrew et al.
1999; Hasbani et al. 1998
; Polischuk et
al. 1998
), and the swelling of mitochondria (Aitken et
al. 1999
; Andrew et al. 1999
) have been
correlated with increase in light scattering.
In the present study, IOS was used to measure light transmittance
through a brain stem slice preparation that contains a functional network generating ongoing rhythmic activity (Smith et al.
1991). We aimed to monitor changes within distinct regions
containing functional networks such as the nucleus hypoglossus and the
periambigual region, which is involved in respiratory rhythm generation
and pattern formation. We investigated changes of the IOS during
normoxia, but also during hypoxia and disturbances of osmotic pressure. Specifically, we focused on the mechanisms that contribute to the
hypoxic IOS-response such as glutamate release and blockade of the
Na+, K+ pump. Finally, we
examined the effects of drugs, acting on KATP channels. These channels are modulated by intracellular ATP levels (Haller et al. 1999
) and thus directly couple cellular
metabolism to modulation of membrane permeability and neuronal
excitability. KATP channels are believed to
modulate the respiratory activity (Pierrefiche et al.
1996
) and to play a vital role in the mechanisms protecting
against hypoxic cell damage (Krishnan et al. 1995
; Mironov et al. 1998
; Murphy and Greenfield
1991
). Our data indicate that neuronal activity together with
activity-dependent ion transportation and presumably mitochondrial
swelling contribute essentially to the IOS.
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METHODS |
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Slice preparations
Experiments were performed on the medullary slice preparation
from neonatal mouse (Mironov et al. 1998; Smith
et al. 1991
). The preparation contains a functional respiratory
network that generates ongoing rhythmic activity. All animals were
housed, cared for, and killed in accordance to the recommendations of the European Commission (No. L 358, ISSN 0378-6978), and protocols were
approved by the Committee on Animal Research, Göttingen University. The brain stem-spinal cord was isolated in ice-cold artificial cerebrospinal fluid (ACSF, composition listed below), and a
single transverse 700-µm-thick slice containing the
pre-Bötzinger complex was cut from the brain stem, transferred to
the recording chamber, and mounted on the stage of an upright
microscope (Axioscop FS, Zeiss). The slice was fully submerged in a
continuously flowing ACSF (28°C, 40-50 ml/min) and gassed with
carbogen (95% O2-5% CO2).
To prevent diffusional loss of dissolved gases, the perfusing solution
was delivered to the experimental chamber via stainless steel tubing.
The respiratory rhythm was established at an elevated extracellular
K+ concentration of 8 mM. One hypoglossal (XII)
rootlet was sucked into a blunt capillary for extracellular recording
of rhythmic respiratory discharges. XII activity was amplified
5,000-10,000 times, band-pass (0.25-1.5 kHz) filtered, rectified, and
integrated (Paynter filter with a time constant of 50-100 ms). This
integrated version of nerve activity typically has an amplitude of 0.1 mV. However, as the amplitude is quite variable and depends on the leak
conductance of the suction electrode, a relative calibration was made
assuming a control peak amplitude of 1 to describe temporal changes of
nerve activity during individual experiments.
All drugs were added directly to the bath reservoir and arrived at the
experimental chamber after a delay of 8-12 s. Drug wash out was
achieved by perfusing 400-500 ml fresh solution containing the same
[K+]o. To induce hypoxic
conditions in the slice, the bubbling gas mixture was changed from
carbogen to 95% N2 and 5%
CO2. The oxygen level was measured with
oxygen-sensitive electrodes (Diamond Electro-Tech, Ann Arbor, MI) as
described previously (Mironov et al. 1998). PO2 electrodes were placed 100 ± 25 (SE) µm below the slice surface, into a region where
inspiratory neurons were located. Fifteen to 20 s after exchanging
oxygen in the perfusing solution by nitrogen, extracellular
PO2 changed from 232 ± 39 to 6 ± 4 mmHg and then remained constant (n = 15, P < 0.05).
Imaging techniques
For IOS measurements, slices were transiently transilluminated
using a tungsten lamp that was controlled by a voltage-regulated power
supply (Zeiss). Near-infrared illumination was obtained using a
highpass filter, which cut off light below 780 nm wavelength. Water
immersion objectives were used to avoid light scattering at the
air/tissue interface, which otherwise would have distorted the IOS
significantly (Kreisman et al. 1995). Objective lenses with different magnifications (×2.5, ×10, ×63) of an upright
microscope were used to obtain images of the whole slice or single
cells. Images were collected with a charge-coupled device (CCD) camera (Princeton Instruments) at rates of up to 1 frame/s. In the
experiments, we followed the procedure described by Andrew and
MacVicar (1994)
. First, control images were recorded, areas of
interest were defined, and averages of the transmitted light were used
as control values, To. The data were
transformed into relative changes, according to IOS =
T/To. Images were
presented using a pseudocolor intensity scale.
For measuring single-cell volume (Fig.
1C),
inspiratory neurons, i.e., neurons that discharged rhythmic bursts of
action potentials in synchrony with hypoglossal (XII) nerve activity,
were filled with mag-fura-2 via the patch pipettes and illuminated with
a monochromator (Till Photonics, Planegg, Germany) with the ultraviolet (UV) light of 346-nm wavelength, which corresponds to the isosbestic point of mag-fura-2 (Raju et al. 1989).
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Solution and drugs
ACSF contained (in mM) 128 NaCl, 3 KCl, 1.5 CaCl2, 1.0 MgSO4, 21 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, pH adjusted to 7.4 with NaOH. Solutions with variable K+-concentration were obtained by replacing NaCl with KCl. Intracellular solution used for whole cell recording contained (in mM) 120 K+-gluconate, 15 NaCl, 2 MgCl2, 10 HEPES, 0.5 Na2ATP, 1 CaCl2, 3 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and sometimes 100 µM mag-fura-2. The pH was adjusted to 7.4 with KOH. Solution osmolarity ranged from 285 to 290 mosM. HMR1098 was kindly provided by Aventis Pharma Deutschland GmbH (Frankfurt, Germany). All other reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany).
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RESULTS |
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IOS during cell swelling
Figure 1A shows the infrared image of the preparation at low magnification. IOS measurements focused on two regions that reveal rhythmic respiratory activity, i.e., the periambigual region (pa) and the nucleus hypoglossus (nh) as defined by rectangles in Fig. 1A. The relative changes in IOS in these regions during hypoxia are displayed in Fig. 1B using a pseudocolor scale (n = 11). In both regions, the IOS revealed a pronounced increase within 4 min after oxygen depletion and recovered completely after 15 min of reoxygenation.
Individual cells were observed to swell during hypoxia (Fig. 1C). Cell volume changes were monitored in identified inspiratory neurons loaded with mag-fura-2 (n = 3). The dye was excited at its isosbestic point for Ca2+ binding (346 nm) to eliminate the contribution of the hypoxic rise in Ca2+ to the fluorescence signal. The cell contours were defined from corresponding cell masks obtained by setting a threshold light intensity filter (right-hand side of Fig. 1C). The bottom panel represents the difference between the mask obtained after 4 min of hypoxia and that of control. Thus it indicates the area of volume increase of the cell as highlighted by pink (positive) color.
The IOS changed during various maneuvers that induced changes in
respiratory activity. Figure 2 shows
simultaneous measurements of the respiratory output as indicated in
hypoglossal nerve activity (XII) and the corresponding IOS obtained
from the nucleus hypoglossus and the periambigual region (gray traces).
Increase of K+ concentration within the external
bath solution from 3 to 9 mM (Fig. 2A) resulted in the
appearance of rhythmic respiratory activity, which was accompanied by a
significant rise of IOS. The IOS changes were significantly larger in
the nucleus hypoglossus as compared with the changes in the
periambigual region indicating a differential effect of ongoing
neuronal and metabolic activity. On re-superfusion with low
[K+] solution (3 mM), IOS values returned to
baseline levels. The IOS was also reversibly changed during variations
in osmolarity of the perfusion solution (Fig. 2B). In slices
exposed to a hyperosmotic solution (+15 mosM) obtained by
supplementation of mannitol, the IOS declined, whereas there was a rise
in IOS in hyposmotic medium (15 mosM). In both cases the changes of
IOS were comparable in size in both regions and returned to control
levels within 15 min after restoration of isosmotic conditions. Thus as
expected, osmotic cell swelling caused an increase in light
transmittance, whereas shrinkage resulted in a decrease.
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Hypoxic IOS response
Hypoxia leads to an initial augmentation of respiratory
activity followed by a secondary depression superimposed on a slowly developing DC signal (Cherniack et al. 1970;
Richter et al. 1991
) (Fig. 2C). The IOS rose
during both phases of hypoxia even though the rate of IOS rise
decreased with time. In some cases the IOS leveled off. As previously
reported (Volker et al. 1995
) metabolic poisoning with
cyanide elicits responses similar to hypoxia (Fig. 2D) and
occludes subsequent hypoxic reactions. Respiratory activity and IOS,
however, recovered fully after reoxygenation or wash out of KCN.
There were important differences in the IOS responses obtained from the two brain stem regions. During the initial phase of hypoxia and after changing extracellular K+, the latency was shorter, and the amplitude as well as the rate of IOS rise were larger in the nucleus hypoglossus as compared with the periambigual region (Fig. 2, A, C, and D). However, there were no such regional differences after changing the osmolarity of the perfusion solution (Fig. 2B).
Hypoxia induces augmented release of excitatory and inhibitory
neurotransmitters and neuromodulators (Richter et al.
1999), which might contribute to the observed hypoxic IOS
responses. The principal excitatory neurotransmitter involved in the
early phase of hypoxia is glutamate, which activates
N-methyl-D-aspartate (NMDA) and
kainate/
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptor subtypes (Choi 1993
; Schurr et al.
1995
). Thus to simulate initial hypoxic responses, NMDA and
kainate were applied to the slice preparation, which resulted in a
marked, but reversible increase in IOS similar to the hypoxic IOS
response. The effects were blocked by the specific antagonists
2-amino-5-phosphonovaleate (AP-5) and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Fig.
3, A and B).
Glutamate application led to a transient rise of the IOS (Fig.
3C). Adenosine, which is released during hypoxic depression (Richter et al. 1999
), induced a transient decrease of
the IOS (Fig. 3D).
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When the glutamate uptake blocker
L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC)
was added, the IOS baseline level decreased, and consecutive periods of
anoxia elicited only diminished IOS responses (Fig. 3E). On
wash out, the IOS returned to control levels, and the hypoxic IOS
response recovered. To further investigate the correlation between IOS
and neurotransmitter release, we applied bafilomycin A1, a potent and
specific blocker of the vacuolar-type (V-type) ATPase, which
eliminates the driving force for the uptake of glutamate, serotonin,
and GABA into synaptic vesicles (Moriyama and Futai
1990; Zhou et al. 2000
). Again we observed a
reversible decrease of the IOS as well as a partial blockade of the
hypoxic IOS response (Fig.
4A).
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Another source of the hypoxic IOS increases might be cell swelling due
to ATP depletion and depression of
Na+/K+-ATPase activity in
the plasma membrane, which leads to accumulation of intracellular
Na+ and loss of intracellular
K+. To test this assumption the
Na+/K+-ATPase blocker
ouabain was applied. On ouabain application (50-100 µM), the
respiratory activity displayed a hypoxia-like reaction, but the IOS
baseline decreased (Fig. 4B). In the presence of ouabain, hypoxic stimuli elicited only 1-2 sequential IOS responses until the reaction of both the respiratory output and IOS were completely suppressed and did not recover even after 1 h of wash out. The final IOS response exhibited a higher amplitude and a delayed recovery
on reoxygenation. The ouabain-induced decrease of IOS could be caused
by cell shrinkage due to Ca2+ entry and
subsequent activation Ca2+-activated
K+ channels (Alvarez-Leefmans et al.
1994; Smith et al. 1993
). To test this
hypothesis, Ca2+ channels were blocked with
Cd2+ before ouabain application. Indeed this
resulted in a pronounced rise in IOS, which was transient and followed
by a prolonged IOS decrease (Fig. 4C).
An important mechanism of volume regulation in glial cells is the
Na+/K+/2Cl-
cotransporter, which mediates the electroneutral uptake of ions (Haas 1994; Kimelberg et al. 1986
) and
thus contributes to cell volume regulation (Baba 1992
;
Chen et al. 1992
; Hoffmann 1992
; Walz 1992
). Blockade of the
Na+/K+/2Cl
cotransporter by furosemide should lead to fall of intracellular Na+, K+ and
Cl- concentrations followed by water efflux and
thus shrinkage of glial cells. Indeed we observed a decrease in the IOS
after furosemide application, which was reversible after 60-80 min
wash out. The hypoxic responses of IOS and respiratory activity
remained unmodified (Fig. 4D), indicating that the
Na+/K+/2Cl
cotransporter does not directly affect hypoxic responses of neuronal activity.
Blocking neuronal activity with tetrodotoxin (TTX) completely suppressed respiratory activity and slowly decreased IOS (Fig. 4E). The hypoxic IOS responses became progressively smaller, but they were not completely suppressed even after cessation of rhythmic output activity.
IOS response following application of KATP channel drugs
Normoxic and hypoxic respiratory activities are effectively
modulated by KATP channels (Mironov et al.
1998; Pierrefiche et al. 1996
). Therefore we
tested the contribution of KATP channels to the
IOS responses by applying channel-specific drugs, such as
glibenclamide, tolbutamide (blockers), diazoxide, and pinacidil (openers) under normoxic conditions. High concentrations of such KATP channel-directed drugs affected the size of
integrated XII bursts as exemplified in Fig.
5A. Application of the channel
opener diazoxide (300-500 µM, n = 7) led to a
decrease of the area of integrated hypoglossal burst activity by
21.2 ± 10.3%, whereas tolbutamide (300-500 µM,
n = 7) and glibenclamide (30-50 µM,
n = 7) resulted in an increase by 15.9 ± 12.8%
and 6.4 ± 6.2%, respectively (Student's t-test,
P < 0.05). We expected that the IOS would rise on
application of KATP channel blockers due to
enhancement of neuronal activity and cell swelling, and that it would
correspondingly decline on application of KATP
channel activators. The results, however, were variable. Both the
applications of the agonist diazoxide (n = 28) and the
blocker glibenclamide (n = 23) resulted in an increase
in IOS in approximately one-half of the tests, but in a decrease in the
remainder trials. Individual responses seemed to be specific for a
given slice preparation in that they could be reproduced over a time
span of several hours. These findings indicate that the mechanisms
underlying generation of the IOS involve also processes other than
neuronal or glial swelling.
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One additional process might be mitochondrial swelling as
KATP channels expressed in mitochondrial
membranes (Hu et al. 1999; Wang and Ashraf
1999
) are also affected by diazoxide and glibenclamide. To test
this possibility, comparable metabolic conditions were established by
10 min of hypoxia followed by the application of KATP channel-directed drugs (Fig.
5B). In this case, the IOS started to rise during hypoxia
and then consistently rose further when the channel opener diazoxide
was applied or decreased after application of the channel blocker
glibenclamide in all tests performed (n = 8). When
HMR1098, which exclusively blocks plasmalemmal
KATP channels, was applied (Fig. 5C),
a reversible increase in IOS was observed in all slices both
during normoxia (n = 5) and hypoxia (n = 3). The involvement of mitochondria was also
verified by applying a mitochondrial uncoupler, carbonylcyanide
m-chlorophenylhydrazone, CCCP. Figure 5D illustrates how
CCCP application led to an initial increase in IOS, which then turned
into a decrease toward levels well below control (n = 3). The latter effect manifested severe damage of cells, as the
respiratory output was irreversibly lost.
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DISCUSSION |
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We used the IOS to monitor metabolic changes in neurons during hypoxia, the application of glutamate receptor agonists and KATP channel blockers or openers. The measurements were complicated by the fact that processes other than cell volume changes appeared to contribute significantly to IOS, necessitating a more detailed investigation of the origin of IOS in the rhythmic slice preparation.
Changes in cell volume
It is commonly accepted that any change in intracellular and/or
extracellular osmolarity induce changes in cell volume (Andrew and MacVicar 1994; Lipton 1973
). In this work,
changes in osmolarity also modulated the IOS in the rhythmic brain stem
slice preparation, verifying that part of the IOS reflects cell volume
changes. This finding was substantiated by analyses of identified
inspiratory neurons, which exhibited a pronounced and reversible
increase in volume in parallel with the IOS rise during hypoxia. The
effect was similar to that described for hippocampal neurons in slices (Kreisman and LaManna 1999
; Turner et al.
1995
).
Regional differences in IOS
There are significant differences between neuronal systems
indicating differences in metabolic demands and/or protective
processes. The measurements of faster and larger IOS changes in
hypoglossal nucleus are consistent with the observations in previous
studies (Haddad and Donnelly 1990; O'Reilly et
al. 1995
; Pierrefiche et al. 1997
), that
hypoglossal motoneurons have a low tolerance to hypoxia. In contrast to
hippocampal neurons and other brain stem neurons, such as respiratory
or dorsal vagal neurons, hypoglossal neurons exhibited a pronounced
anoxic depolarization in response to O2
deprivation (Donnelly et al. 1992
; O'Reilly et
al. 1995
; Pierrefiche et al. 1997
;
Richter et al. 1991
, 1992
). A larger anoxic depolarization in hypoglossal neurons would be expected to lead
to a higher degree of cell swelling due to a larger influx of ions.
Indeed, comparison of respiratory neurons with hypoglossal motoneurons
revealed pertinent differences in the hypoxic IOS responses. After
changes in bath osmolarity (±15 mosM), however, the IOS displayed no
regional diversity, which indicates that passive water movement imposed
by osmolarity changes affects the volume of all cell populations in a
similar manner.
Mechanisms underlying the hypoxic IOS-response
As mentioned above, hypoxia has been observed to elicit a
pronounced IOS response that is in part due to cell swelling. The most
likely mechanisms underlying hypoxic cell swelling are
Na+, Ca2+, and
Cl influxes (Haddad and Donnelly
1990
; Mercuri et al. 1994
) and failure of the
Na+/K+ pump following ATP
depletion (Calabresi et al. 1995
; Le Corronc et
al. 1999
). After reoxygenation, the IOS recovered, indicating the restoration of the original cell volume. Important factors contributing to such regulatory volume decrease are
Na+/K+ pump activity
(Basavappa et al. 1998
; Fraser and Swanson
1994
) or parallel operation of Cl
and
K+ channels, allowing efflux of
K+ and Cl
(Hoffmann 1992
; Lippmann et al. 1995
).
The various factors causing cell swelling and thus contributing to the
hypoxic IOS increase were investigated by application of
neurotransmitters, neurotransmitter uptake blockers, and blockade of
Na+/K+ pumping.
EFFECT OF NEUROTRANSMITTERS ON IOS.
Our experiments verified that processes that enhance neuronal activity,
such as glutamate receptor activation, produce a transient increase in
IOS, whereas adenosine (an agent that depresses spontaneous neuronal
activity) transiently decreases IOS. The transient nature of the effect
can be explained by immediate neurotransmitter reuptake. During hypoxia
a prolonged accumulation of neurotransmitters and neuromodulation is
expected due to impairment of uptake processes and catabolic enzymes in
neurons and glial cells (Haddad and Jiang 1993;
Neubauer et al. 1990
). During the early hypoxic
augmentation phase, predominantly glutamate and GABA are released,
which is followed by a delayed release of adenosine and serotonin
during hypoxic respiratory depression (Richter et al.
1999
). Consequently, we assume that a glutamate-induced IOS
increase contributes to the early hypoxic rise in IOS, whereas an
adenosine-mediated fall contributes to the decreasing slope of the
hypoxic IOS change that was observed during the depressive phase of
hypoxia. This assumption is corroborated by the finding that a
reduction in glutamate release, either induced by the glutamate uptake
blocker PDC (Fig. 3E) or vacuolar ATPase inhibitor
bafilomycin A1 (Fig. 4A), resulted in a general decline of
IOS and a partial blockade of the hypoxic IOS response.
NA+/K+-PUMP AND NEURONAL
ACTIVITY.
It is generally accepted that depression of
Na+/K+ pump activity leads
to cell swelling due to accumulation of intracellular Na+ (Buckley et al. 1999;
Shimizu and Nakamura 1992
), thereby causing an increase
in IOS similar to that observed during hypoxia. Surprisingly, blocking
of Na+/K+ pump activity
with ouabain did not mimic the hypoxic IOS response, but
rather markedly decreased IOS baseline levels. This is consistent with
the observations in a number of studies (Alvarez-Leefmans et al.
1994
; Smith et al. 1993
) that ouabain induces
cell shrinkage rather than cell swelling. The authors suggested that
ouabain induces a transient elevation of
[Ca2+]i, which in turn
activates a K+ efflux through
Ca2+-activated K+ channels,
leading to water loss and thus cell shrinkage. Indeed, the effect could
be transiently reversed when Ca2+ entry was
blocked with CdCl2 (Fig. 4D). This
suggests that ouabain-induced cell shrinkage contributes to the IOS. In
addition, complimentary factors other than changes in total cell volume
might participate in the ouabain-induced IOS response (Buchheim
et al. 1999
; Muller and Somjen 1999
). For
instance, ouabain-induced excitotoxicity, as described by
Zeevalk and Nicklas (1996)
, might lead to irreversible changes in dendritic morphology, denominated dendritic
beading (Andrew et al. 1999
; Polischuk et
al. 1998
). Previously it has been suggested that dendritic
beading reflects damage of dendrites, e.g., following a prolonged
exposure to hypoxia and/or to high levels of NMDA (Hori and
Carpenter 1994
; Park et al. 1996
). Dendritic beading is expected to result in a decrease in IOS (Andrew et al. 1999
; Polischuk et al. 1998
). Such action
could also account for the irreversibility of the ouabain action.
Involvement of mitochondrial KATP channels in IOS generation
Another mechanism associated with IOS is swelling of cytoplasmatic
organelles, such as mitochondria (Aitken et al. 1999), which has long been known to result of hypoxia and/or ischemia (Aitken et al. 1999
; Allen et al. 1989
;
Lazriev et al. 1980
; Vladimirov Iu and Kogan
1981
). Swelling of mitochondria is accompanied by a decrease in
light scattering (Mar 1981
) or light absorbance (Stoner and Sirak 1969
). Flow cytometry analysis also
showed that swelling of individual mitochondria leads to a decrease in
light absorbance (Beavis et al. 1985
;
Macouillard-Poulletier de et al. 1998
), indicating that
mitochondria behave as light-scattering objects that affect the IOS in
the same way as the whole cell (i.e., swelling leads to an increase in
IOS, shrinkage to a decrease).
An indication for the contribution of mitochondrial swelling to
IOS generation was observed when KATP channel
drugs were applied. KATP channels contribute to
IOS in a dual manner. 1) KATP channels regulate the excitability of respiratory neurons (Pierrefiche et
al. 1997), and therefore application of
KATP channel blockers/activators leads to a
rise/fall in the IOS, as the neurons are depolarized/hyperpolarized. In
the present experiments, the blocker of plasmalemmal
KATP channels, HMR1098, exhibited this behavior.
2) A secondary effect of the drugs seems to target at other
structures, e.g., mitochondrial KATP
(mitoKATP) channels. Activation of
mitoKATP channels with diazoxide was previously
observed to induce depolarization of the mitochondrial membrane
potential (Grimmsmann and Rustenbeck 1998
; Gross
and Fryer 1999
; Holmuhamedov et al. 1998
), which
normally induces mitochondrial swelling. KATP
antagonists reversed this effect (Garlid et al. 1997
).
To test for the dual effects of KATP channels,
slices were first exposed to a prolonged period of hypoxia prior to
drug application to induce maximal activation of plasmalemmal KATP channels. Under such conditions, application
of KATP-directed drugs induced IOS responses that
could be attributed primarily to changes in
mitoKATP channel activity (fall after
glibenclamide, rise after diazoxide). The results were remarkably
reproducible, indicating that the effects of plasmalemmal and
mitochondrial KATP channels indeed differ and can
be distinguished in this way. The contribution of mitochondrial IOS
signals was further verified through applying the uncoupler of
mitochondrial oxidative phosphorylation, CCCP, which depolarizes
mitochondria and induces mitochondrial swelling (Minamikawa et
al. 1999
). Such findings indicate that during prolonged
hypoxia, KATP channel blockers and openers act on
mitochondrial KATP channels rather than on
plasmalemmal KATP channels. A similar finding,
i.e., that glibenclamide is much less potent in inhibiting plasmalemmal
KATP channels after metabolic poisoning, was
previously observed in pancreatic
-cells (Mukai et al.
1998
).
In conclusion, the hypoxic IOS response can be interpreted as a result of several distinct mechanisms as illustrated in Fig. 6. First, there is a component that is related to cell swelling, and, second, there are mechanisms such as dendritic beading or mitochondrial swelling. Cell swelling can be attributed to anoxic depolarization due to hypoxic release and accumulation of neurotransmitters and neuromodulators, while Na+/K+ pump activity is inhibited. The processes acting through Na+ fluxes can be subdivided into two components, one being mediated through voltage-regulated and TTX-sensitive Na+ channels and another that is TTX insensitive.
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ACKNOWLEDGMENTS |
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We thank N. Hartelt and J. Huhnold for expert technical assistance.
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FOOTNOTES |
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Address for reprint requests: M. Haller, Physiologisches Institut, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Gottingen, Germany (E-mail: mirjam{at}neuro-physiol.med.uni-goettingen.de).
Received 31 July 2000; accepted in final form 14 March 2001.
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REFERENCES |
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