Electrophysiological Recordings and Calcium Measurements in Striatal Large Aspiny Interneurons in Response to Combined O2/Glucose Deprivation

Antonio Pisani,1 Paolo Calabresi,1 Diego Centonze,1 Girolama A. Marfia,1 and Giorgio Bernardi1,2

 1Clinica Neurologica, Dipartimento di Neuroscienze, Università di Roma Tor Vergata, 00133 Rome;  2IRCCS Ospedale S. Lucia, 00173 Rome, Italy


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). 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.

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; Silver et al. 1997; Tymianski et al. 1994).

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, 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.

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 >= 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.

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, omega -agatoxin TK, omega -conotoxin GVIA, and omega -conotoxin MVIIC were from Alomone Labs (Israel).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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,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 (right-arrow).

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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Fig. 2. Pharmacological characterization of LA interneurons. A: bath-applied oxotremorine (300 nM, 2 min) hyperpolarizes and blocks spontaneous firing activity of an LA interneuron (bottom). Top: small concomitant reduction in the basal level of [Ca2+]i. B: in the same cell 20 min after wash-out of oxotremorine, baclofen (10 µM, 2 min) also induces an hyperpolarization and blocks firing discharge. Concomitantly, a decrease of the [Ca2+]i level is observed. Note the different level of basal [Ca2+]i in the somatic and dendritic region ( and black-triangle, respectively), due to the spontaneous firing activity of the cell. C: 20 min after wash-out of baclofen, combined O2/glucose deprivation causes an hyperpolarization and cessation of spontaneous spiking activity. Simultaneously, a large increase in [Ca2+]i is detected in the dendrites. RMP, -62 mV.



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Fig. 3. Time course of the changes induced by O2/glucose deprivation. Graphs show the time course of the effects of O2/glucose deprivation on both [Ca2+]i and membrane potential. Top: note the different Ca2+ signals between the soma () and the dendrites (black-triangle). Each data point represents the mean of >= 8 independent observations. Bottom: pseudocolor ratio images of [Ca2+]i changes observed in a cell (1, dendritic region, 2, somatic area) during O2/glucose deprivation. Color bar codes for [Ca2+]i changes.

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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Fig. 4. Lack of effect of TTX on O2/glucose-deprivation-induced changes. A, top: [Ca2+]i elevations induced by 5 min O2/glucose deprivation in the soma () and in the dendrites (black-triangle). Bottom: recording trace of the simultaneous membrane hyperpolarization. B: bath-applied TTX (1 µM) does not affect basal [Ca2+]i level nor does it modify the changes observed in 5 min O2/glucose deprivation. RMP, -66 mV.

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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Fig. 5. Effects of K+ channels blockers. A: plot and the trace show the [Ca2+]i rise (in the soma, , and in the dendrites, black-triangle) and the membrane hyperpolarization caused by 5 min O2/glucose deprivation. B: in the presence of glibenclamide (100 µM), the membrane hyperpolarization is prevented (bottom), whereas the [Ca2+]i rise is unaffected (top). RMP, -64 mV. C: in this cell, 4 min O2/glucose deprivation induces a large elevation in [Ca2+]i and a membrane hyperpolarization in control condition. D: bath-applied charybdotoxin (300 nM) does not modify both the basal and the elevations in [Ca2+]i observed during O2/glucose deprivation. However, the presence of charybdotoxin induces spontaneous activity and blocks the "ischemic" membrane hyperpolarization. RMP, -62 mV.

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 omega -agatoxin TK (P type), 1 µM omega -conotoxin GVIA (N type), and 1 µM omega -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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Fig. 6. Voltage-gated Ca2+ channels contribute to the response to O2/glucose deprivation. A, top: [Ca2+]i rise caused by 4 min O2/glucose deprivation in the soma () and in the dendrites (black-triangle). Bottom: electrophysiological trace of the simultaneous membrane hyperpolarization. B: after incubation with a cocktail of voltage-dependent Ca2+ channel blockers (20 µM nifedipine, 20 nM omega -agatoxin TK, 1 µM omega -conotoxin GVIA, and 1 µM omega -conotoxin MVIIC) for >10 min, both the [Ca2+]i rise (top) and the membrane hyperpolarization (bottom) were significantly reduced. RMP, -67 mV.



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Fig. 7. Summary plots of the pharmacological data. A: histograms showing the pharmacological effects of TTX, glibenclamide (GLIB), charybdotoxin (CBTX), and a cocktail containing 20 µM nifedipine, 20 nM omega -agatoxin TK, 1 µM omega -conotoxin GVIA, and 1 µM omega -conotoxin MVIIC on dendritic [Ca2+]i () and membrane potential () changes caused by O2/glucose deprivation. See text for somatic [Ca2+]i changes. Data are means, expressed as percentage of control, ±SE. B: these data were obtained from experiments performed without bis-fura 2 in the recording electrode. Both BAPTA and barium significantly reduce the membrane hyperpolarization () caused by O2/glucose deprivation (mean ± SE).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society