1Clinica Neurologica,
Pisani, Antonio,
Paolo Calabresi,
Diego Centonze,
Girolama A. Marfia, and
Giorgio Bernardi.
Electrophysiological recordings and calcium
measurements in striatal large aspiny interneurons in response to
combined O2/glucose deprivation. The effects of
combined O2/glucose deprivation were investigated on large
aspiny (LA) interneurons recorded from a striatal slice preparation by
means of simultaneous electrophysiological and optical recordings. LA
interneurons were visually identified and impaled with sharp
microelectrodes loaded with the calcium (Ca2+)-sensitive
dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a
membrane hyperpolarization coupled to a concomitant increase in
intracellular Ca2+ concentration
([Ca2+]i). Interestingly, this
[Ca2+]i elevation was more pronounced in
dendritic branches rather than in the somatic region. The
O2/glucose-deprivation-induced membrane hyperpolarization
reversed its polarity at the potassium (K+) equilibrium
potential. Both membrane hyperpolarization and
[Ca2+]i rise were unaffected by TTX or by a
combination of ionotropic glutamate receptors antagonists,
D-2-amino-5-phosphonovaleric acid and
6cyano-7-nitroquinoxaline-2,3-dione. Sulfonylurea glibenclamide, a
blocker of ATP-sensitive K+ channels, markedly reduced the
O2/glucose-deprivation-induced membrane hyperpolarization
but failed to prevent the rise in [Ca2+]i.
Likewise, charybdotoxin, a large K+-channel (BK) inhibitor,
abolished the membrane hyperpolarization but did not produce detectable
changes of [Ca2+]i elevation. A combination
of high-voltage-activated Ca2+ channel blockers
significantly reduced both the membrane hyperpolarization and the rise
in [Ca2+]i. In a set of experiments performed
without dye in the recording electrode, either intracellular
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
or external barium abolished the membrane hyperpolarization induced by
O2/glucose deprivation. The hyperpolarizing effect on
membrane potential was mimicked by oxotremorine, an M2-like muscarinic
receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an
increase but rather to a decrease of the basal
[Ca2+]i. Furthermore glibenclamide did not
reduce the oxotremorine- and baclofen-induced membrane
hyperpolarization. In conclusion, the present results suggest that in
striatal LA cells, O2/glucose deprivation activates a
membrane hyperpolarization that does not involve ligand-gated
K+ conductances but is sensitive to barium, glibenclamide,
and charybdotoxin. The increase in [Ca2+]i is
partially due to influx through voltage-gated high-voltage-activated Ca2+ channels.
The high energy demand of the brain results from the requirement
to maintain the ionic gradients and the resting membrane potential
(Erecinska and Silver 1989 Compelling evidence has demonstrated that excessive levels of
intracellular Ca2+ concentration
([Ca2+]i) represent a key step in the cascade
of events leading to cell death in course of both ischemic and
excitotoxic insult (Choi 1990 The aim of the current investigation was to study the responses of LA
interneurons to conditions limiting ATP generation. To attain this, we
used a combined experimental approach to monitor simultaneously both
the electrophysiological and [Ca2+]i changes.
In particular, we focused on the characterization of the membrane
hyperpolarization; establishing the time course, magnitude, and
possible source of the increase in [Ca2+]i;
and comparing the O2/glucose-deprivation-induced electrical and ionic changes with those obtained by activation of ligand-gated conductances.
Preparation and maintenance of the slices
Male Wistar rats (20-30 postnatal day) were used for the
experiments. Preparation and maintenance of the slices have been previously described in detail (Calabresi et al. 1995 Electrophysiological and optical recordings
For electrophysiological recordings sharp microelectrodes were
filled with a KCl (2 M) solution. For combined optical and electrical
recordings, the tip of the recording electrode was filled with a
solution of 1 mM bis-fura-2 (hexapotassium salt, Molecular Probes,
Leiden, The Netherlands) in 1 M KCl. The shank of the recording
electrode was backfilled with a 2 M KCl solution. After cell
impalement, cells were loaded with bis-fura-2 by injecting 0.1- to
0.5-nA negative current for 15-30 min, during which the dye was
allowed to diffuse to the dendritic branches. In some experiments the
recording electrode was filled with
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA, 200 mM); therefore bis-fura 2 was not added.
An Axoclamp 2A amplifier (Axon Instruments) was used for
electrophysiology. Traces were displayed on an oscilloscope and stored on a digital system. In the single-electrode voltage-clamp mode, switching frequency was 3 kHz. The headstage signal was monitored continuously on a separate oscilloscope. In the experiments with bis-fura-2 into the recording electrode, we did not measure the changes
in cell input resistance because voltage steps do interfere with basal
[Ca2+]i levels.
Fluorescence of bis-fura-2 was excited by epi-illumination with light
provided by a 75 W Xenon lamp band-pass filtered alternatively at 340 or 380 nm. Emission light passed a barrier filter (500 nm) and was
detected by a CCD camera (Photonic Science, UK). Pairs of 340 and 380 nm images were acquired at intervals of 12 s and analyzed off-line
with a software (IonVision, ImproVision, UK) running on PowerMac 8100. In the experiments performed with oxotremorine and baclofen, images
were aquired each 4 s.
Calibration of bis-fura-2 signals
Ratio images were calculated from pairs of 340 and 380 nm images
corrected for background fluorescence (measured from regions free of
dye fluorescence). Ratio values were calculated from regions that
included either the cell bodies or distal dendritic regions, defined as
"regions of interest," i.e., those pixels that exhibit Values in the text and in the figures are expressed as means ± SE. Student's t-test was used for statistical analysis.
Drug source and application
Drugs were bath-applied by switching the solution to one
containing known concentrations of drugs. Drugs solutions entered the
chamber within ~20 s after turning on a three-way tap.
D-2-amino-5-phosphonovaleric acid (D-APV) and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Tocris
Cookson (Bristol, UK). TTX, glibenclamide, BAPTA, barium, baclofen,
nifedipine, and oxotremorine were from Sigma (St. Louis, MO).
Charybdotoxin, Electrophysiological and morphological identification of LA
interneurons
Data were obtained from 78 large aspiny interneurons. The cells on
the surface were identified visually through the objective and impaled
with sharp microelectrodes. The electrophysiological features of this
striatal neuronal subtype have been described previously
(Calabresi et al. 1998a
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). Hence even brief
disruptions of energy supply cause irreversible damage to central
neurons. The neuronal vulnerability is region specific, and the
striatum is among the most susceptible areas to ischemic insult
(Pulsinelli 1985
). Indeed within the mammalian striatum,
neuronal subtypes express a differential vulnerability. Projecting
cells, GABAergic medium-sized neurons, with spiny dendrites, are very
sensitive both to ischemia and to excitotoxic damage (Beal et
al. 1986
; Francis and Pulsinelli 1982
;
Pulsinelli 1985
). On the basis of electrophysiological,
morphological, and pharmacological criteria, three different classes of
interneurons have been identified (Kawaguchi 1993
;
Kawaguchi et al. 1995
; Wilson et al.
1990
). Among these, a small percentage, 2-3%, is represented
by large aspiny (LA) interneurons. These cells are known to be
cholinergic and give rise to the intrinsic striatal cholinergic
innervation (Bolam et al. 1984
), exerting a strong
influence on striatal circuitry both in physiological and pathological
conditions. In fact, LA interneurons have been shown to be of crucial
importance for procedural and associative learning tasks
(Graybiel et al. 1994
). In addition, cholinergic
interneurons are spared selectively in pathological conditions
involving the striatum exclusively, such as Huntington's chorea
(Ferrante et al. 1985
) or partially, such as brain
ischemia (Francis and Pulsinelli 1982
). Despite the
experimental evidence of such functional relevance, these cells have
been difficult to approach because of their rarity. Therefore the
reasons behind the selective vulnerability/resistance of striatal
neuronal subtypes during energy deprivation are still largely unknown.
Previous studies have shown that striatal medium spiny cells respond to hypoxia (Calabresi et al. 1995
) and aglycemia
(Calabresi et al. 1997
) with a large, progressive
membrane depolarization, whereas in striatal LA interneurons, aglycemia
induces a membrane hyperpolarization. This membrane hyperpolarization
was prevented by barium and reduced by bathing the slices in a
low-Ca2+-containing solution (Calabresi et al.
1997
). Several reports have shown that in different brain areas
neurons hyperpolarize in response to anoxia, aglycemia, or a
combination of both, through the activation of K+
conductances. These conductances have been identified mainly as
ATP-sensitive K+ currents in hippocampal (Mourre et
al. 1989
), nigral (Jiang et al. 1994
), cortical
(Jiang and Haddad 1997
; Luhmann and Heinemann 1992
), and dorsal vagal neurons (Trapp and Ballanyi
1995
). However, in isolated mouse sensory neurons
(Duchen 1990
), in hippocampal neurons (Leblond
and Krnjevic 1989
), and in locus coeruleus neurons (Murai et al. 1997
), a significant contribution to the
membrane hyperpolarization during energy depletion has been attributed to Ca2+-activated K+ conductances.
; Silver et al.
1997
; Tymianski et al. 1994
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,
1997
; Pisani et al. 1998
). Briefly, animals were
killed under ether anesthesia by cervical dislocation, the brain was
removed, and coronal slices (180- to 200-µm thick), containing cortex
and striatum, were cut from tissue blocks with a vibratome. Slices were
maintained at 34°C in an oxygenated solution (see composition further
in paragraph), for ~30 min. A single slice then was transferred into
a recording chamber, mounted on the stage of an upright microscope
(Axioskop FS, Zeiss), equipped with a ×60, 0.90 n.a.
water-immersion objective (LUMPlan FI, Olympus), and fully submerged in
a continuously flowing Krebs solution (33°C, 3 ml/min) gassed with
95% O2-5% CO2. The composition of the
solution was (in mM) 126 NaCl, 2.5 KCl, 1.3 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and
18 NaHCO3. The "ischemic" medium was a glucose-free
solution equilibrated with a 95% N2-5% CO2
gas mixture. Glucose was replaced with saccharose to balance
osmolarity. Complete replacement of the medium in the chamber took
45-60 s.
20-30%
of maximal specific fluorescence. Ratiometric measurements were
converted into ion concentration values according to Grynkiewicz et al. (1985)
. The calibration parameters
Rmin and Rmax were
obtained in situ by bathing perforated (ionomycin) cells in
Ca2+-free (1 mM EGTA) and in 1 mM
Ca2+-containing solution. The apparent
Kd values for bis-fura-2 was estimated by
measuring fluorescence ratios obtained from solution containing
bis-fura-2 and known concentrations of free Ca2+ trapped
between two coverslips spaced by pieces of coverslips and imaged with
the water immersion objective. This in vitro Kd was consistent with the Kd estimated from
perforated cells.
-agatoxin TK,
-conotoxin GVIA, and
-conotoxin
MVIIC were from Alomone Labs (Israel).
RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,b
; Kawaguchi
1993
; Kawaguchi et al. 1995
; Wilson et
al. 1990
): these cells had a relatively depolarized resting
membrane potential (
62 ± 5 mV), low threshold for spike
discharge, and high input resistance. As shown in Fig. 1B, current pulses evoked a
firing activity that was limited by a strong spike frequency adaptation
and by a pronounced afterhyperpolarization. Hyperpolarizing current
pulses evoked a prominent sag conductance (Ih,
Fig. 1C, a), which is known to be cesium sensitive
(Jiang and North 1991
). These are unique properties of
this striatal cell subtype. The sag conductance opposes pronounced
hyperpolarization that would keep the cell too far from the firing
threshold, whereas the strong spike afterhyperpolarization prevents the
cell from firing continuously. These cells also express a
K+ conductance, of the A type, which is believed to
contribute to a great extent to the modulation of the slow, repetitive
firing activity of LA interneurons (Song et al. 1998
).
In some cases, 0.1- to 0.3-nA negative bias current was used to prevent
spike discharge.
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Fig. 1.
Characterization of a striatal large aspiny (LA) interneuron.
A: fluorescence image of an LA interneuron filled with
bis-fura 2 (average of 256 frames, 380 nm). Note the large somatic
region and 2-4 main dendrites. Bar = 25 µm. B:
current-evoked action potential discharge in an LA cell induces rapid
accomodation. Note also the pronounced afterhyperpolarization.
C: combined O2 and glucose deprivation (5 min) induces an hyperpolarization with a reduction in the input
resistance of the recorded cell as measured by current steps;
bottom: current steps at higher speed (400 pA, 2 s)
in control condition (a) and at peak of the response to
O2/glucose deprivation (b). D:
current-voltage relationship of the O2 and glucose
deprivation response. Reversal potential, obtained from 11 cells is
106 ± 3.3 mV (
).
The dye fluorescence allowed further morphological
identification. As previously described (Kawaguchi 1993;
Kawaguchi et al. 1995
), these cells had a large soma
(~25-50 µm), fusiform shape, two to four primary dendritic
branches (Fig. 1A).
Pharmacological characterization of LA interneurons
LA interneurons have been shown to be cholinergic and to generate
the intrinsic striatal cholinergic innervation (Bolam et al.
1984). Recently, muscarine and muscarinic M2-like receptor agonists have been shown to hyperpolarize LA interneurons that were
positive for choline acetyltransferase immunoreactivity
(Calabresi et al. 1998b
). Therefore to strengthen the
electrophysiological and morphological identification, we tested
oxotremorine, a muscarinic M2-like receptor agonist on the recorded
cells. As shown in Fig. 2A,
bath-applied oxotremorine (300 nM, 2 min) blocked the spontaneous activity and hyperpolarized the cell. This effect was accompanied by a
small, transient decrease of the basal
[Ca2+]i (Fig. 2A, bottom). It
should be reminded that the whole population of the recorded cells did
not show spontaneous firing activity; however, also in these cells
oxotremorine could cause an hyperpolarization and a decrease of the
basal [Ca2+]i. Yet in spontaneously spiking
cells, the basal [Ca2+]i was slightly higher
(+12 ± 1.3 nM) both in soma and dendrites. Baclofen (10 µM, 2 min) also hyperpolarized the recorded cells and reduced the resting
[Ca2+]i (Fig. 2B). Similarly
O2/glucose omission caused a membrane hyperpolarization,
but this was accompanied by a large increase in
[Ca2+]i (Fig. 2C). The response of
this cell was representative for the overall population as demonstrated
by the average time-course values of membrane potential and
[Ca2+]i obtained from bis-fura-2 loaded cells
(Fig. 3) To exclude the possible
interference by baclofen and oxotremorine with the responses to
O2/glucose omission, we performed this pharmacological
identification in eight cells after the exposure to a solution lacking
O2 and glucose, and no difference was detected.
Glibenclamide (100 µM), an ATP-sensitive K+ channel
blocker, failed to block the oxotremorine-induced hyperpolarization (n = 6, data not shown).
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Effects of combined O2/glucose deprivation
The effects of O2/glucose deprivation on LA cells were
time dependent. We chose an exposure time of 4-5 min, which is
considered sufficient in in vivo models of ischemia to induce permanent
cell damage (Pulsinelli 1985; for review, see
Hara et al. 1993
). In addition, after 5 min of perfusion
with a O2/glucose-deprived solution, both the membrane
hyperpolarization and the [Ca2+]i levels were
fully reversible. Figure 3 shows the time course of the electrical and
[Ca2+]i changes induced by
6 min
O2/glucose deprivation. As shown, the early hyperpolarizing
response and the rise in [Ca2+]i occurred
simultaneously and developed gradually. A prominent [Ca2+]i elevation was observed mainly in the
dendrites, whereas in the somatic region the
[Ca2+]i rise was much smaller. Each time
point was the average of at least eight single observations ±SE. Data
pooled at 2, 3, 4, 5, and 6 min were statistically significant
(P < 0.01).
As shown in Fig. 1D, the current-voltage relationship shows
that the reversal potential during O2/glucose deprivation
was very close to the K+ equilibrium potential, 106
mV ± 3.3 mV. The reversal potential of the
O2/glucose-deprivation-induced hyperpolarization was
calculated by measuring the steady-state current induced by voltage
steps of long duration (2 s) and progressively increasing amplitude from the holding potential before and during O2/glucose
omission. The trace in Fig. 1C shows the fall in input
resistance in course of O2/glucose deprivation (compare a
and b). By changing the external K+ concentration from 2.5 mM (control medium) to a solution containing 5 mM KCl, the reversal
potential was shifted toward more positive potential, according to the
Nernst equation (
90 ± 2.6 mV; n = 4; not shown).
Lack of effect of TTX and glutamate receptor antagonists on O2/glucose deprivation
To evaluate the possible role of TTX-sensitive sodium channels and transmitter release on the O2/glucose-induced changes, TTX (1 µM) was bath applied 5-10 min before the solution lacking O2/glucose. TTX did not affect the resting membrane potential per se nor the basal [Ca2+]i. Yet, compared with the response observed in control conditions (Fig. 4A) in the presence of TTX, the membrane hyperpolarization and the [Ca2+]i elevation during O2/glucose omission were unaltered (Figs. 4B and 7A; 101 ± 8% of control of dendritic [Ca2+]i, P > 0.01; 118 ± 8.6% of control of somatic [Ca2+]i, P > 0.05; 111 ± 10.7% of control of Vm, P > 0.05; n = 5).
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To examine whether the rise in [Ca2+]i was mediated by ionotropic glutamate receptors, control responses to O2/glucose omission were compared with those obtained in a solution containing 50 µM D-APV and 10 µM CNQX. These antagonists block N-methyl-D-aspartate (NMDA) and AMPA types of ionotropic glutamate receptors. Bath application of a combination of 50 µM D-APV and 10 µM CNQX (10-15 min) did not affect the resting membrane potential and the resting [Ca2+]i, nor did they induce significant modifications of the membrane hyperpolarization and of the [Ca2+]i increase during perfusion with a medium deprived of O2 and glucose (measured at 5 min) (98.6 ± 7% dendritic [Ca2+]i, P > 0.05; 89.6 ± 10.3% somatic [Ca2+]i, P > 0.05; 88.1 ± 8.3% Vm, P > 0.01; n = 4; data not shown).
Effect of blockade of K+ channels and of BAPTA
Bath-application of the ATP-dependent K+ channel blocker glibenclamide (30-100 µM, 10-15 min) did not alter either the membrane potential or the [Ca2+]i at rest (Fig. 5B). However, compared with control response (Fig. 5A), in the presence of 100 µM glibenclamide, the membrane hyperpolarization observed during O2/glucose deprivation was reduced markedly (Figs. 5B and 7A; 11 ± 3% of control Vm, P < 0.01, n = 7). Conversely, the [Ca2+]i was not affected (Figs. 5B and 7A; 96 ± 14% of control dendritic [Ca2+]i, P > 0.05, n = 7; 101 ± 8.9% of control somatic [Ca2+]i, P > 0.01, n = 7).
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Perfusion with 300 nM charybdotoxin, a BK channel blocker, did not affect the basal [Ca2+]i (Fig. 5D); in addition the [Ca2+]i elevation caused by O2/glucose deprivation was not modified (Figs. 5D and 7A; 91 ± 16% dendritic [Ca2+]i, P > 0.05, n = 4; 89 ± 8% somatic [Ca2+]i, P > 0.01, n = 4). However, 1-2 min after the onset of charybdotoxin application, a sustained spontaneous activity of the recorded cell was detected (Fig. 5D), probably because these conductances operate at rest in these cells. In comparison with control response, in 300 nM charybdotoxin the O2/glucose-deprivation-induced membrane hyperpolarization was prevented fully (Figs. 5D and 7A; 8 ± 1.2% of control Vm, P < 0.01, n = 4). Finally, in a set of experiments performed without bis-fura-2 in the recording electrode, we tested the nonspecific blocker of K+ channels barium. Bath-applied barium (1 mM, 15 min) strongly reduced the membrane hyperpolarization caused by O2/glucose omission (Fig. 7B, 11 ± 2% Vm, n = 4, P < 0.01).
In three experiments, recording electrodes were filled with BAPTA (200 mM), chelator of intracellular Ca2+. BAPTA was allowed to diffuse into the cell by injecting negative current (0.1-0.3 nA, 20-30 min). Proof that BAPTA was acting effectively was provided by the significant reduction of the afterhyperpolarization after the current-induced spike discharge and consequent reduced spike frequency adaptation (results not shown). In the presence of intracellular BAPTA, the membrane hyperpolarization generated by O2/glucose omission was significantly reduced (Fig. 7B; 18 ± 3% of control Vm, P < 0.05, n = 3).
Role of voltage-dependent Ca2+ channels
To identify the possible source responsible for the
[Ca2+]i elevation, we performed two sets of
experiments using nickel, to assess the role of low-voltage-activated
(LVA) Ca2+ currents and cadmium as an aspecific blocker of
voltage-gated Ca2+ channels. Bath-applied 50 µM nickel
did not modify either the resting values of both membrane potential and
[Ca2+]i or the response to
O2/glucose omission (n = 3; data not
shown), ruling out a possible involvement of LVA Ca2+
currents. In the second set of experiments, a few minutes after the
onset of perfusion with 100 µM cadmium, a significant increase in the
fluorescence signal was detected (n = 5, not shown).
This effect may be due to the high affinity of cadmium for the
fluorescent dye used in the present work. In fact, fura-2
fluorescence has been used to investigate the cadmium uptake into
glial cells (Hinkle et al. 1992). Therefore, we used a
cocktail of HVA Ca2+ channel blockers: 20 µM
nifedipine for L-type HVA channels, 20 nM
-agatoxin TK (P
type), 1 µM
-conotoxin GVIA (N type), and 1 µM
-conotoxin
MVIIC (Q type) (Wheeler et al. 1994
). Bath application (10-15 min) of this cocktail did not alter the resting membrane potential of the recorded cells nor did it change the basal
[Ca2+]i (Fig.
6B); however, compared with
the control response (Fig. 6A) in course of
O2/glucose deprivation both the membrane hyperpolarization and the dendritic [Ca2+]i increase were
significantly reduced (Figs. 6B and
7A; 69 ± 5.78% of
control Vm; 68.6 ± 1.33% of control
dendritic [Ca2+]i; P < 0.05, n = 6). Conversely, somatic
[Ca2+]i rise was unaffected; it is likely
that a dominant distribution of HVA channels on the dendritic branches
compared with the somatic region accounts for this effect.
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DISCUSSION |
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The results of the present study accomplished three main goals: they defined the time course and magnitude of the electrical and ionic events related to O2/glucose deprivation in LA interneurons; the K+ conductances involved in the membrane hyperpolarization have been shown to be both glibenclamide-sensitive and Ca2+-dependent, the [Ca2+]i, increase is partially due to influx through HVA Ca2+ channels; and both the membrane hyperpolarization and the [Ca2+]i elevation are TTX insensitive and do not depend on the activation of ligand-gated conductances.
Features of the membrane hyperpolarization induced by O2/glucose deprivation
A membrane hyperpolarization in response to O2 and/or
glucose deficiency has been described in several central neurons and attributed mainly to the opening of ATP-sensitive K+
channels. This response to metabolic stress has been interpreted as a
defense mechanism by which the cell can slow down energy consumption
(Haddad and Jiang 1993).
In the present work, we found a contribution of both
glibenclamide- and charybdotoxin-sensitive K+ conductances.
Likewise, in rat locus coeruleus neurons, the hypoxia-induced outward
current was inhibited either by tolbutamide or by charybdotoxin (Murai et al. 1997). It should be noted that the authors
reported that the tolbutamide-sensitive K+ current
component decreased time dependently. A similar observation also was
obtained from substantia nigra dissociated neurons exposed to hypoxia
(Jiang et al. 1994
). This might be consistent with a
putative temporal correlation between the opening of the two conductances. In fact, low O2 levels might transiently
activate an ATP-sensitive K+ conductance; the simultaneous
[Ca2+]i rise then would activate the large,
charybdotoxin-sensitive Ca2+-dependent K+
channels. Moreover, it has been demonstrated recently that in neocortical neurons, the activation of a large ATP-dependent, glibenclamide-sensitive K+ conductance required the
presence of micromolar concentrations of Ca2+ (Jiang
and Haddad 1997
). Finally, both the time course of
[Ca2+]i rise and the reduction of membrane
hyperpolarization by both HVA Ca2+ channel blockers and
BAPTA during O2/glucose deprivation support the involvement
of a Ca2+-dependent K+ conductance in the
generation of the electrical changes in LA interneurons.
Role of neurotransmitters
LA cholinergic interneurons give rise to the intrinsic striatal
cholinergic network (Bolam et al. 1984). Muscarinic
M2-like receptor agonists have been shown to hyperpolarize cholinergic LA cells (Calabresi et al. 1998b
). We used this
pharmacological effect as a further means for the identification of LA
cells. In addition to the reported muscarinic inhibition of
voltage-gated Ca2+ currents (Yan and Surmeier
1996
), this membrane hyperpolarization might account for the
muscarinic inhibitory control of acetylcholine release within the
striatum. Accordingly, during application of oxotremorine, a selective
M2-like muscarinic receptor agonist, we observed a decrease in the
basal [Ca2+]i compared with the large
[Ca2+]i rise occurring in course of
O2/glucose depletion. A similar effect was observed with
baclofen; noticeably, this GABAB receptor agonist also has
been shown to inhibit Ca2+ currents (Harayama et al.
1998
). Hence these data support the involvement of different
mechanisms underlying the transmitter-induced membrane
hyperpolarization compared with the electrical changes observed during
O2/glucose deprivation.
Recently we have shown that LA interneurons respond to activation of
both NMDA and AMPA glutamate receptors with a membrane depolarization.
The sensitivity of LA interneurons to these agonists was, nevertheless,
lower than that observed for medium spiny neurons (Calabresi et
al. 1998a). It might be argued that because our recording
electrode was in the cell soma, dendritic elevation in
[Ca2+]i induced by NMDA and AMPA receptor
activation could account for the [Ca2+]i rise
caused by O2/glucose omission. However, NMDA and AMPA receptor antagonists, bath-applied 10-15 min before the perfusion with
an O2/glucose-lacking solution failed to block the membrane hyperpolarization and [Ca2+]i elevation
caused by energy deprivation. Although seemingly contradictory, these
data are consistent with the hypothesis that glutamate-induced
neurotoxicity is different from that observed in course of
O2/glucose depletion (Friedman and Haddad
1993
; Silver et al. 1997
). Furthermore, the lack
of effect by TTX seems to rule out the involvement of TTX-sensitive
glutamate release.
Role of [Ca2+]i rise
Ion gradients are very sensitive to impairment of energy
metabolism (Erecinska and Silver 1989; Silver et
al. 1997
). Experimental work supports the involvement of
Ca2+ as the main responsible for cell damage in the course
of both excitotoxic and ischemic damage (Choi 1990
;
Friedman and Haddad 1993
; Silver et al.
1997
; Tymianski et al. 1994
). We found a
significant increase of dendritic [Ca2+]i
occurring simultaneously with the membrane hyperpolarization. The
blockade of this K+ current by both glibenclamide and
charybdotoxin, however, did not affect the
[Ca2+]i elevation. This lack of effect by
both glibenclamide and charybdotoxin on the
[Ca2+]i signal is conceivable, assuming that
these drugs would act downstream at the channel level. In fact, the
significant reduction of both the [Ca2+]i
increase and the membrane hyperpolarization by a cocktail of HVA
Ca2+ channel blockers strengthens the idea that an initial
[Ca2+]i rise is necessary for the opening of
a BK channel. Moreover, the inhibition of the membrane potential
changes by intracellular BAPTA also supports the Ca2+
dependency of the alterations observed in course of
O2/glucose omission. Indeed, it remains to be established
to what extent different routes contribute to the rise in
[Ca2+]i. We have shown that LVA channels do
not seem to be involved in this response, considering the lack of
effect by nickel (Magee and Johnston 1995
;
Todorovic and Lingle 1998
). Conversely, the blockade of
HVA channels (L, N, P, and Q type) (Wheeler et al. 1994
)
provided evidence for an involvement of these Ca2+ channels
in the generation of the [Ca2+]i signal. At
present we cannot rule out a possible contribution of R-type channels.
Also the release of Ca2+ from intracellular stores might
account for the remaining component of the [Ca2+
]i signal in course of O2/glucose deprivation.
Noticeably, the [Ca2+]i elevation occurring
in the dendritic area was, by far, larger than the somatic one. It
might be hypothesized that the Ca2+-buffering systems in
the soma can handle the Ca2+ overload better than do
dendrites (Blaustein 1988). This could be related to the
expression and distribution of Ca2+-binding proteins.
Nevertheless, LA cholinergic interneurons appear devoid of any known
Ca2+-buffering protein (Bennett and Bolam
1993
; Kita et al. 1990
). Moreover,
immunocytochemical studies examining the distribution of
inositol-1,4,5-triphosphate (IP-3) and ryanodine receptors in the rat
striatum demonstrated that only ryanodine receptor labeling was present
in LA cholinergic interneurons, whereas IP-3 labeling was virtually
absent in these cells (Martone et al. 1997
). Further
work is required to address the contribution of
Ca2+-buffering systems in the
[Ca2+]i elevation in conditions of energy limitation.
Functional implications
LA cholinergic interneurons are thought to exert an associative
task, thereby integrating glutamatergic inputs arising from the cortex
and thalamus with the dopaminergic inputs originating from the
substantia nigra (Kawaguchi et al. 1995).
Electrophysiological recordings performed in primates in vivo show that
"tonically active" striatal cells closely resemble the activity
of in vitro identified LA cholinergic interneurons (Graybiel et
al. 1994
). During behavioral conditioning and learning tasks,
these cells respond in a temporally related manner, influencing the
pattern of electrical activity of other striatal neuronal subtypes.
Even in course of pathological conditions, such as Huntington's
disease and brain ischemia, these cells have been shown to possess
peculiar features, being selectively spared, whereas medium spiny cells are lost (Ferrante et al. 1985
; Francis and
Pulsinelli 1982
).
The reasons for the unique behavior of these cells are not clear yet. Thus the understanding of the mechanisms underlying the behavior of these cells in conditions of energy depletion appears of primary importance to clarify both physiological and pathophysiological characteristics of these neurons.
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ACKNOWLEDGMENTS |
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We thank M. Tolu for excellent technical assistance, Dr. P. Gubellini for helpful comments, and G. Bonelli for help in preparing the illustrations.
This work was supported by National Research Plan on Neurobiologic Systems-Signal Transduction Technologies, by Biomed Grant BMH4-97-2215 to P. Calabresi, and by a Ministero Universitá e Ricerca Scientifica e Tecnologica grant to G. Bernardi.
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FOOTNOTES |
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Address for reprint requests: A. Pisani, Clinica Neurologica, Dipartimento Neuroscienze, Università Tor Vergata, via di Tor Vergata 135, 00133 Rome, Italy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 August 1998; accepted in final form 7 January 1999.
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REFERENCES |
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