1Fondazione Santa Lucia, Istituto di Ricovero e Cura a Carattere Scientifico; 2Clinica Neurologica, Universita' di Roma "Tor Vergata"; and 3Clinica Neurologica, Universita' di Roma "La Sapienza," 00179 Rome, Italy
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ABSTRACT |
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Marinelli, Silvia,
Mauro Federici,
Patrizia Giacomini,
Giorgio Bernardi, and
Nicola B. Mercuri.
Hypoglycemia Enhances Ionotropic But Reduces Metabotropic
Glutamate Responses in Substantia Nigra Dopaminergic Neurons.
J. Neurophysiol. 85: 1159-1166, 2001.
It is
widely accepted that energy deprivation causes a neuronal death that is
mainly determined by an increase in the extracellular level of
glutamate. Consequently an excessive membrane depolarization and a rise
in the intracellular concentration of sodium and calcium are produced.
In spite of this scenario, the function of excitatory and inhibitory
amino acids during an episode of energy failure has not been studied
yet at a cellular level. In a model of cerebral hypoglycemia in the rat
substantia nigra pars compacta, we measured neuronal responses to
excitatory amino acid agonists. Under single-electrode voltage-clamp
mode at 60 mV, the application of the ionotropic glutamate receptor
agonists N-methyl-D-aspartate,
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate, and
the metabotropic group I agonist (S)-3,5-dihydroxyphenilglycine (DHPG)
produced reversible inward currents in the dopaminergic cells. In
addition, an outward current was caused by the superfusion of the
metabotropic GABAB agonist baclofen. Glucose
deprivation enhanced the inward responses caused by each ionotropic
glutamate agonist. In contrast, hypoglycemia depressed the DHPG-induced
inward current and the baclofen-induced outward current. These effects
of hypoglycemia were reversible. To test whether a failure of the
Na+/K+ ATPase pump could
account for the modification of the agonist-induced currents during
hypoglycemia, we treated the midbrain slices with strophanthidin (1-3
µM). Strophanthidin enhanced the inward currents caused by glutamate
agonists. However, it did not modify the
GABAB-induced outward current. Our data suggest
that glucose deprivation enhances the inward current caused by the
stimulation of ionotropic glutamate receptors while it dampens the
responses caused by the activation of metabotropic receptors. Thus a
substantial component of the augmented neuronal response to glutamate,
during energy deprivation, is very likely due to the failure of
Na+ and Ca2+ extrusion and
might ultimately favor excitotoxic processes in the dopaminergic cells.
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INTRODUCTION |
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The progress of neuronal
dysfunction and damage during energy deprivation is a complex process
that includes presynaptic and postsynaptic mechanisms (Auer and
Siesjo 1988; Martin et al. 1994
). Two main
events have been described when energy levels are reduced: an increased
release of excitatory amino acids (EAA) and a reduced concentration of
intracellular ATP, which leads to diminished Na+/K+-ATPase activity
(Benveniste et al. 1984
; Erecinska and Silver 1989
; Hansen 1985
; Lees 1991
;
Roettger and Lipton 1996
). It is well accepted that the
excessive stimulation of EAA receptors associated with metabolic
inhibition hampers the recovery of
[Na+]i and
[Ca2+]i loads and
facilitates cell death (Cebers et al. 1998
; Lees 1991
; Monyer et al. 1989
; Novelli et al.
1988
; Rose et al. 1998
; Rothman et al.
1987
). However, the neuronal vulnerability caused by energy
deprivation might not only result from an excess of extracellular
glutamate and aspartate that stimulates NMDA and/or AMPA/kainate
receptors (Choi 1988
) but also from an impaired function of metabotropic responses. For instance, a deficient hyperpolarization mediated by GABA metabotropic receptors might not be able to counteract the harmful ischemia-induced membrane depolarization produced by
glutamate receptors superactivation. In addition, the neuronal damage
resulting from energy failure might not be simply dependent on the
increased extracellular levels of EAAs. In fact, the reduced metabolic
state might aggravate neuronal depolarizations to glutamate and
aspartate, and this can influence outcome.
In spite of the importance of the excitatory and inhibitory processes in the pathogenic sequences caused by energy deprivation, there are only few studies that have examined the changes in responses to excitatory and inhibitory amino acids under a reduced metabolic condition.
Considering that the early electrophysiological events caused by
hypoxia and hypoglycemia have been extensively studied in the
dopaminergic neurons of substantia nigra pars compacta (Guatteo et al. 1998a,b
; Hauser et al. 1991
; Jiang
et al. 1994
; Marinelli et al. 2000
;
Mercuri et al. 1994a
,b
; Stanford and Lacey
1995
; Watts et al. 1995
), that these cells
possess well-characterized postsynaptic responses to ionotropic and
metabotropic glutamate receptor agonists, and to the
GABAB agonists (Lacey et al. 1988
; Mercuri et al. 1992a
,b
, 1993
), and that a selective
toxicity of these cells occurs during impairment of energetic
metabolism and activation of NMDA receptors (Marey-Semper et al.
1995
), we used single-electrode voltage-clamp recordings from
dopaminergic neurons of the rat mesencephalon maintained in vitro to
examine changes in glutamatergic and GABAergic responses during glucose
removal. We report that hypoglycemia enhances the cellular responses
caused by the activation of ionotropic glutamate receptors and
depresses the responses mediated by second messengers.
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METHODS |
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Tissue preparation
Wistar rats (150-300 g) were killed under deep halothane
anesthesia. All efforts were made to minimize animal suffering. The brain was rapidly removed from the skull, and horizontal slices (300 µm thick) of the ventral midbrain were cut by a vibratome (Mercuri et al. 1995). A single slice containing the
substantia nigra and the ventral tegmental area was transferred to a
recording chamber, immobilized with titanium grids, and perfused at a
rate of 2.5 ml/min, with a solution maintained at 35°C and oxygenated with a mixture of 95%
O2-CO2 5%
O2. The standard solution contained (in mM) 126 NaCl, 2.5 KCl, 1.2 NaH2PO4,
1.2 MgCl2, 2.4 CaCl2, 10 glucose, and 19 NaHCO3, pH of 7.4.
Intracellular recording techniques
The recording electrodes (Clark 1-1.5 mm, thick wall), pulled
by a Flaming-Brown horizontal puller (Sutter Instruments, Novato, CA),
were filled with 2 M KCl and had a tip resistance of 30-80 M.
Membrane voltage and current signals were recorded using an amplifier
(Axoclamp-2A, Axon Instruments, Foster City, CA). During the
single-electrode voltage-clamp procedures, the amplifier headstage was
monitored on a separate oscilloscope to ensure correct operation of the
switch clamp: switching frequency was 3-4 kHz, 30% duty cycle. The
signals were displayed on a pen recorder and on a digital oscilloscope
or digitized by use of an A/D converter (Digidata 1200, Axon
Instruments) and saved in a computer with the Axotape software (Axon
Instruments) for off-line analysis. To obtain I-V plots,
voltage commands (40-100 ms, between
110 and
40 mV) were delivered
before and during the application of the agonists in the presence of
TTX (1 µM) and barium (300 µM). The I-V curves in the
presence of N-methyl-D-aspartate (NMDA) were
also done in magnesium-free solutions. The substantia nigra pars
compacta was visually identified as the region caudal to the medial
terminal nucleus of the accessory optic tract using a dissecting microscope.
Induction of hypoglycemia and application of drugs
To induce hypoglycemia, the control solution was substituted
with aglycemic artificial cerebrospinal fluid (ACSF, 0 mM glucose) saturated with 95% O2-5%
CO2. In some experiments, equimolar mannitol (10 mM) was replaced with glucose. The drugs were bath-applied at a known
concentration. Drug solutions entered the recording chamber no later
than 20 s after turning a three-way tap. Complete replacement of
the medium in the chamber took 90 s. The following drugs were
used: -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 10 µM) and (S)-3,5-dihydroxyphenilglycine (DHPG, 50 µM) from Tocris
Cookson, NMDA (50 µM), kainic acid (KA, 50 µM), strophanthidin
(1-3 µM), tetrodotoxin (TTX, 1 µM), and dopamine hydrochloride
(10-30 µM) from Sigma. Baclofen (30 µM) was obtained from Roche.
Data analysis
Numerical data were expressed as means ± SD. Student's t-test for paired observations was used to compare the data. P < 0.05 was considered significant. The areas of the currents induced by ionotropic and metabotropic agonists have been calculated with Microcal Origin-Analysis/Calculus/Integrate-program running on an IBM computer.
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RESULTS |
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Properties of the dopaminergic cells
The present study is based on intracellular recordings from 86 "principal" dopaminergic neurons of the rat substantia nigra pars
compacta. These cells were identified by their location in the slice
and their well-defined electrophysiological and pharmacological characteristics (Grace and Ohn 1989; Lacey et al.
1988
, 1989
; Mercuri et al. 1995
).
Effects of ionotropic glutamate agonists
Under single-electrode voltage-clamp condition (60 mV, holding
potential), transient application of NMDA (50 µM for 15-30 s)
induced a rapidly developing inward current (Fig.
1A). This inward current could
be repeatedly produced on the same neuron. The average inward current
caused by NMDA was 261 ± 63 pA (n = 8). The total
charge of the NMDA current was 37 ± 8 pC (n = 8, Fig. 2A). The rapid
application AMPA (10 µM for 10-20 s) also produced a rapidly
developing inward current that washed quickly (Fig. 1). This inward
response was consistently caused on each application of AMPA. The
average inward current caused by 10 µM AMPA was 678 ± 121 pA
(n = 8). The total charge caused by AMPA was 69 ± 8 pC (n = 8, Fig. 2A).
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The rapid superfusion of kainate (50 µM for 15-30 s) caused an inward current of 191 ± 48 pA (n = 6). This type of response could be repeatedly evoked on the same neuron. The total charge induced by kainate was 31.5 ± 11 pC (n = 6, Fig. 2A).
Responses to metabotropic agonists
The rapid superfusion of DHPG (50 µM for 20-30 s), a Group I metabotropic agonist, produced an inward current of 107 ± 20 pA (n = 6) that could be consistently reproduced on the same cell (Fig. 1B). The total current area caused by DHPG was 15.5 ± 3 pC (n = 6, Fig. 2B).
The rapid superfusion of baclofen (30 µM for 15-20 s) determined an outward current of 395 ± 52 pA (n = 5, Fig. 1B). The outward response to this GABAB agonist could be consistently reproduced on the same cell. The total charge caused by baclofen was 46.3 ± 16 pC (n = 5, Fig. 2B).
Effects of hypoglycemia on the agonist-mediated responses
The perfusion of a glucose-free ACSF for 15-20 min produced
a slowly developing outward current (Marinelli et al.
2000) that was 96 ± 8 pA (n = 19) at 15 min and 304 ± 33 pA (n = 19) at 20 min. We routinely tested the effects of the
agonists after 20 min of glucose depletion. The inward current caused
by NMDA was greatly augmented by hypoglycemia (Fig. 1A). The
peak current was 329 ± 59% (n = 8, P < 0.05) of control. In addition we observed an
increase of the duration of the current. Thus the inward area was
932 ± 242% (n = 8, P < 0.05) of
control (from 37 ± 8 to 345 ± 68 pC). Similar results
were obtained with AMPA and kainate (Fig. 1A). In fact, the
peak AMPA and kainate currents were 157 ± 30% P < 0.05 (n = 8) and 344 ± 96% P < 0.05 (n = 6) of control, respectively. In addition,
the total AMPA current was 453 ± 105% of control
(n = 8; from 69 ± 8 to 313 ± 89 pC;
P < 0.05) and the total kainate current was 251 ± 44% of control (n = 6; from 31.5 ± 11 to
78 ± 28 pC; P < 0.05; Fig. 2A). After
having examined the effects of hypoglycemia on the glutamate-evoked
ionotropic currents, we also tested the effects on the metabotropic
current caused by the group I agonist DHPG. After 20 min of glucose
depletion, the inward charge caused by DHPG was depressed by 64 ± 8% of control (n = 6; from 15.5 ± 3 to 5.6 ± 1.5 pC; P < 0.05; Figs. 1A and 2B). A depressant effect of hypoglycemia on the
agonist-induced current was also observed when we tested the effect of
the metabotropic GABAB agonist baclofen (Fig.
1B). In fact, the outward charge caused by baclofen was
reduced by 70.4 ± 4%, of control (n = 5; from
46.3 ± 16 to 13.7 ± 3.9 pC; P < 0.001, Fig. 2B). All the enhancing or depressing effects of
hypoglycemia on the agonist-induced currents (3 cells for each agonist)
were reversible and were also observed in the presence of TTX (1 µM,
which was used to block fast sodium channel and synaptic transmission).
In a series of experiments using the glutamate agonists, we also
applied barium (300 µM) to reduce either the potassium conductance
increase caused by energy deprivation (Guatteo et al.
1998a
; Marinelli et al. 2000
; Mercuri et
al. 1994b
) or the outward current that follows the application
of the glutamate agonists (Mercuri et al. 1996
); under
the treatment with this divalent cation, hypoglycemia enhanced NMDA,
AMPA, and kainate responses and reduced the DHPG-induced inward current
(2 neurons for each agonist). In addition, hypoglycemia did not cause
changes of the I-V relationship of the currents caused by
ionotropic (Fig. 3) and metabotropic
agonists (3 neurons for each compound; data not shown).
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Effects of strophanthidin on the agonist-induced currents
To investigate the role of the
Na+/K+-ATPase pump in
determining changes in the agonist-induced currents in the dopaminergic neurons (voltage-clamped at 60 mV), the agonists were tested before,
during, and after the superfusion of the pump inhibitor, strophanthidin
(1-3 µM). The superfusion of this compound initially induced an
inward current (87 ± 35 pA, n = 17), but after
13-15 min, perfusion caused an outward shift of the holding current (109 ± 15 pA, n = 17). In the presence of
strophanthidin, the responses induced by the glutamate agonists were
increased (Fig. 4). In fact, the NMDA-,
AMPA-, KA-, and DHPG-induced inward currents were increased to 555 ± 29% (n = 4, P < 0.05), 394 ± 99% (n = 4, P < 0.05), 266 ± 52% (n = 3, P < 0.05), and
214 ± 109% (n = 4, P < 0.05) of
control, respectively (Fig. 6). Interestingly, while the metabotropic
(DHPG-induced) current was increased, the baclofen-induced current was
not significantly modified (128 ± 58% of control,
n = 4, P = 0.32) in the presence of
strophanthidin (Figs. 5 and
6). In addition, strophanthidin did not
cause a clear-cut change of the I-V relationship during the
effects of the excitatory and inhibitory agonists (2 cells for each
compound; not shown).
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DISCUSSION |
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The major observation of the present study is that a lack of glucose differentially changes the ionotropic and metabotropic responses caused by glutamate agonists in midbrain dopaminergic cells. In fact, the ionotropic-induced inward currents were increased, while the metabotropic inward current were decreased. In addition the metabotropic GABAB-induced outward current was depressed. Altogether, these phenomena could be key elements in the constitution of brain damage during energy deprivation.
The observation that the inward responses caused by the ionotropic
glutamate agonists were potentiated by glucose removal is, at least in
part, consistent with the hypoglycemia induced prolongation of
NMDA-induced inward current previously observed in dissociated
dopaminergic cells of the substantia nigra (Nakashima et al.
1996). In addition, here we show that hypoglycemia not only
prolongs but also increases the peak NMDA inward currents.
Previous studies have already demonstrated that anoxia either
suppresses the NMDA-induced current (Krnjevic et
al. 1989) or does not affect the postsynaptic responses
to AMPA and NMDA (Khazipov et al. 1995
) in hippocampal
neurons. The differences between the present results and those obtained
in the hippocampus might be due to the use of different cell types or
different methods of obtaining energy deprivation (hypoglycemia vs. anoxia).
The basis for the hypoglycemia enhancement of the glutamate
currents is an unbalanced sodium and calcium clearance. In fact, during
the activation of NMDA, AMPA, and KA channels there is an increased
permeability to sodium and calcium. Under normal conditions
(normoglycemic), the
Na+/K+-ATPase extrudes
Na+ and energizes other secondary ion
transporters (e.g.,
Na+/Ca2+ exchange). If a
profound drop in the level of extracellular glucose occurs, the
consequent ATP depletion causes a reduced extrusion of
Na+ and Ca2+ ions from the
intracellular compartment (Haddad and Jiang 1993). It is
important to note that during metabolic inhibition, a relevant component of the [Na+]i
load derives from glutamate-gated Na+ influx
(Auer and Siesjo 1988
; Choi and Rothman
1990
). In addition, an increase in the content of
[Ca2+]i might derive not
only from the activation of calcium permeable glutamate receptors but
also from the reversal of
Na+/Ca2+ exchanger
(Amoroso et al. 1993
; Stys et al. 1991
).
All these events certainly cause a harmful disturbance of ion
regulation (Kiedrowski et al. 1994
), leading to a
reduction of membrane potential, cell swelling, and irreversible damage.
In agreement with the hypothesis that an impaired function of the
Na+/K+-ATPase augments the
cellular responses to excitatory amino acids during hypoglycemia, we
observed that the strophanthidin-induced block of this pump amplifies
the inward currents caused by glutamate agonists. Consistent with the
present results, it has been previously reported that the activity of
the Na+/K+-ATPase pump
regulates the glutamate-induced depolarization of striatal and
hippocampal neurons (Calabresi et al. 1995;
Fukuda and Prince 1992
). However, changes of
postsynaptic glutamate receptor conformation may also be involved
during hypoglycemia.
The reduced glycolysis caused by the lack of energy substrates would be
likely to produce a drop in intracellular ATP and GTP levels,
which would limit the activation of G-protein-mediated events.
Consequently, during hypoglycemia, the activation of metabotropic responses such as those caused by DHPG and baclofen (Tanabe et al. 1998) is reduced. In accordance, the baclofen-induced
currents were not significantly modified by a treatment with
strophanthidin. The fact that the agonist-induced responses along the
explored voltage range was not modified by hypoglycemia or
strophanthidin treatment further suggests that alteration of ion
homeostasis and not of ion reversal potential be involved.
Physiopathological implications
It is widely accepted that the neuronal damage caused by energy
deprivation is highly dependent on the release of excitatory amino
acids (Auer 1986; Choi 1988
;
Siesjo et al. 1988
; Szatkowski and Attwell
1994
; Vornov 1995
), which has negative
consequences, including a sustained membrane depolarization, an
increase in intracellular Na+ and
Ca2+, increase of extracellular potassium, and
activation of reactive oxygen species (Coyle and Puttfarchen
1993
; Dugan et al. 1995
; Guatteo et al.
1998b
; Peng and Greenamyre 1998
; Rose et
al. 1998
; Szatkowski and Attwell 1994
;
Tymianski et al. 1993
; Waxman et al.
1994
).
In particular the dopaminergic cells are highly sensitive to
excitotoxicity and oxidative stress when the energetic metabolism is
impaired (Chan et al. 1994; Jenner et al.
1992
; Marey-Semper et al. 1995
; Shapira
1994
).
The present electrophysiological data suggest that a reduced activity of Na+/K+-ATPase pump, induced by a reduction of energy levels due to glucose deprivation, enhances glutamate ionotropic responses in the dopaminergic cells of the substantia nigra pars compacta. Thus not only an increased release of excitatory amino acids but also amplified postsynaptic responses to glutamate associated to depressed GABAB-mediated hyperpolarizations might contribute to the vulnerability of these neurons during an episode of energy failure.
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ACKNOWLEDGMENTS |
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We thank Drs. Christopher W. Vaughan and Ezia Guatteo for suggestions.
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FOOTNOTES |
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Address for reprint requests: N. B. Mercuri, Experimental Neurology Laboratory, IRCCS Fondazione Santa Lucia, Via Ardeatina 306, 00179 Rome, Italy (E-mail: Mercurin{at}med.uniroma2.it).
Received 21 July 2000; accepted in final form 22 November 2000.
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REFERENCES |
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