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(Stroke. 1996;27:127-133.)
© 1996 American Heart Association, Inc.

Articles

Effects of Cerebral Ischemia on N-Methyl-D-Aspartate and Dihydropyridine-Sensitive Calcium Currents

An Electrophysiological Study in the Rat Hippocampus In Situ

Turgay Dalkara, MD, PhD; Cenk Ayata, MD; Mehmet Demirci, MD; Gül Erdemli, MD, PhD Rüstü Onur, MD, PhD

From the Departments of Neurology (T.D., M.D.) and Pharmacology (C.A., G.E., R.O.), Hacettepe University, Faculty of Medicine, Ankara, Turkey.

Correspondence to Turgay Dalkara, MD, PhD, Department of Neurology, Hacettepe University Hospital, Ankara, Turkey, TR 06100.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Background and Purpose During cerebral ischemia, both promoting and limiting factors are present for activation of the N-methyl-D-aspartate (NMDA) receptor ion channel and the dihydropyridine (DHP)-sensitive Ca2+ channels. We investigated the activity of these channels during ischemia and reperfusion in the rat hippocampus in situ.

Methods Reversible ischemia was induced by bilateral carotid artery ligation. NMDA and BAY K8644 were applied by iontophoresis or pneumatic ejection, and extracellular field potential and resistance changes were recorded from the CA1 region of the rat hippocampus. Resting membrane potentials of the CA1 neurons were also recorded.

Results DC potential shifts produced by NMDA and BAY K8644 were reduced when ischemia depressed the evoked activity more than 50%. They disappeared on total failure of synaptic transmission and recovered during reperfusion. When the evoked activity was depressed less than 50%, DC shifts were greater than their preischemic values; however, BAY K8644-induced potentiation did not reach statistical significance. CA1 neurons were depolarized during ischemia.

Conclusions These data suggest that ischemia severe enough to cause transmission failure inactivates NMDA and DHP-sensitive Ca2+ currents. During less intense ischemia and reperfusion, NMDA and DHP-sensitive Ca2+ channels are functional, and their overactivation may lead to neurotoxicity.


Key Words: calcium channels • cerebral ischemia • hippocampus • N-methyl-D-aspartate • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Increased cytosolic Ca2+ is a major cause of cell damage in cerebral ischemia.1 2 The NMDA receptor ion channel and DHP-sensitive Ca2+ channels are considered among the possible routes of Ca2+ entry to neurons during ischemia.3 4 5 6 However, failure of NMDA receptor antagonists to prevent tissue damage in global ischemia led to the idea that there might be factors limiting NMDA receptor overactivation under ischemic conditions.7 8 9 10 Demonstration of phosphorylation dependency of DHP-sensitive Ca2+ channels and depression of their activity during anoxia also cast some doubt on their role in energy-deficient states.11 12 13 It is likely that ischemia triggers both facilitatory and inhibitory processes acting on these channels, and the overall effect may vary depending on the severity of ischemia. We investigated this possibility by following the activity of these channels during the course of cerebral ischemia and reperfusion. Channel activity was induced by local application of NMDA or the Ca2+ channel agonist BAY K8644 in the rat hippocampus and was monitored by recording population currents and Rt changes.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Humane treatment of experimental animals was strictly applied throughout the housing and experimental periods. The procedures followed were within local institutional guidelines. The experiments were performed on 22 male Swiss albino rats weighing 200 to 300 g. Anesthesia was induced by urethane (1.4 to 1.6 g/kg IP). Both carotid arteries were exposed after a ventral median surgical incision on the neck and suspended with silk sutures. After completion of surgery on the neck, rats were placed in a stereotaxic apparatus. The rectal temperature was maintained at 37°C to 38°C. The heart rate was monitored.

Stimulating electrodes were placed in the fimbria/commissure at the following coordinates: anterior 6.5, lateral 0.5, height +7.0, according to the atlas of Albe-Fessard et al.14 Multibarreled glass microelectrodes were used for recording and iontophoretic and pneumatic administration of drugs. Recording barrels were routinely filled with 3 mol/L NaCl, and the other barrels were filled with N-methyl-DL-aspartate (Sigma Chemical Co; 50 mmol/L in 0.15 mol/L saline, pH 8) or BAY K8644 (Bayer AG; 100 mol/L in 1% ethanol and 0.15 mol/L saline). NMDA was applied by iontophoresis or pressure ejection, BAY K8644 only by pressure. The specificity of the effects observed with these agents was determined by antagonizing their effects by MK-801 (Merck Sharp & Dohme; 40 mmol/L in 0.15 mol/L saline, 15 to 100 nA) and nimodipine (Bayer AG; 50 mmol/L in 1% ethanol and 0.15 mol/L saline, 7 to 15 psi) (Fig 1Down). Pressure application of ethanol (1% in 0.15 mol/L saline) or 0.15 mol/L saline did not produce any effect except a 15% to 25% drop in Rt, possibly due to expansion of extracellular space by volume injection. Positioning of the recording electrode in the CA1 region of the right hippocampus was guided by observation of the characteristic field response evoked by fimbrial/commissural stimulation and the stereotaxic coordinates (anterior 4.0, lateral 2.0 to 2.5, height {approx}2). Extracellular DC potential changes (DC shifts) during drug applications were recorded in addition to the stimulus-evoked activity. The current applied for iontophoresis was automatically balanced with a current having the same magnitude but opposite polarity to minimize the potential shift due to iontophoretic current. Borosilicate, glass microelectrodes without filament filled with 3 mol/L KCl and having tip resistances of 40 to 60 M{Omega} were used for intracellular recordings. RMP was taken as the difference between the potential recorded before and immediately after the cell was penetrated to avoid extracellular DC potential changes and electrode tip potentials, which make the estimation of transmembrane potential difference complicated during long-lasting intracellular recordings and ischemia. Glia were easily identified with their high membrane potential, absence of synaptic potentials, and failure to fire with depolarizing pulses.



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  		Figure 1. A, Nimodipine (NIMO) completely inhibits DC shifts induced by pneumatic ejection of BAY K8644 (BAYK) (7.2 psi, 1 second) in the CA1 region of the hippocampus. BAY K8644 was regularly applied every 2 minutes (dots), and nimodipine was given by pneumatic ejection for 6 minutes (15 psi). A partial recovery was observed 10 minutes after cessation of nimodipine. B, MK-801 (100 nA, 6 minutes) blocks DC shifts induced by iontophoretic ejection of NMDA (70 nA, 10 seconds) in the CA1 region of the hippocampus. NMDA was regularly applied every 2 minutes (dots). A partial recovery was observed 17 minutes after cessation of MK-801. C, The resistance-measuring pulses were recorded at points indicated by arrowheads on tracing B and illustrate the inhibition of NMDA-induced extracellular resistance increase by MK-801.

Cellular swelling or shrinkage due to either ischemia or drug application was monitored by measuring the change in electrical resistance at the tip of microelectrode. A constant current pulse (10 to 50 nA, 10 milliseconds, 1 Hz) was applied between one barrel of the microelectrode and a remote silver plate placed subcutaneously in the neck, and another barrel was used to record the voltage drop, which is directly proportional to the resistance of the tissue between the tip of the voltage barrel and the remote electrode. However, because the current lines converge as they approach the tip of the electrode, the contribution of tissue to the resistance increases as it gets closer to the tip.15 16 17 Therefore, practically, the measured resistance is essentially determined by the tissue immediately surrounding the tip of the microelectrode.

Ischemia was produced by ligation of carotid arteries, which was previously shown to provide a suitable model to study the electrophysiological changes in the rat hippocampus during moderate ischemia.18 19 Briefly, carotid arteries were pulled into glass tubes by using silk sutures to interrupt the carotid blood flow and released for recirculation. First, the ipsilateral carotid was ligated and, if amplitude of the evoked activity was depressed by more than 10%, drugs were tested at this mildly ischemic level. Ischemia was subsequently deepened by ligating the contralateral carotid artery.19 Recirculation was commenced after total failure of the evoked response. In some animals, controlled bleeding was required after bilateral carotid ligation to completely abolish the field response. Since the severity of ischemia was highly variable during the first 20 minutes, possibly depending on the adequacy of collateral circulation, we used the depression of the evoked activity as a measure of the severity of ischemia.

Peak amplitudes of the signals were measured from the baseline. Values are expressed as mean±SEM.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Effects of Ischemia on NMDA Responses
NMDA application by iontophoresis (30 to 70 nA, 10 seconds, n=21) or pressure (6 to 17 psi, 2 to 5 seconds, n=10) in the CA1 region of the hippocampus caused an extracellular negative DC potential shift of 8 to 12 mV (Fig 2Down). This was accompanied by an increase in local Rt (30% to 150%). Both effects were totally reversed by MK-801 (15 to 100 nA) and therefore were attributed to cation influx through the NMDA receptor ion channel and subsequent cellular swelling20 21 22 (Fig 1Up). Responses to NMDA were reversible within 1 minute, and no desensitization was observed with repeated applications of NMDA every 2 minutes. Regular applications were continued during ischemia and reperfusion. On induction of ischemia, NMDA-induced DC shifts were initially increased up to 332% of their preischemic amplitude (Figs 2Down and 3Down and TableDown). NMDA-induced local Rt increases were also enhanced up to 458% of their preischemic values. Potentiation of the NMDA responses was usually seen within the first 2 minutes of ischemia and was associated with less than approximately 40% depression of the evoked field potential. NMDA-induced DC shifts and Rt increases were progressively inhibited as the evoked activity was further depressed. NMDA responses could not be induced after disappearance of the evoked activity even if the amount of NMDA applied was increased (Figs 2Down and 3Down). Both responses to NMDA recovered concurrently with the evoked potential during reperfusion (Figs 2Down and 3Down) and, in three rats, along with spontaneous partial recovery of the evoked activity during ischemia.



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  		Figure 2. A representative experiment showing the effects of ischemia and reperfusion on the evoked activity (top tracings), NMDA-induced DC shifts (middle tracings), and local Rt changes (bottom graphs). Recordings were done in the stratum pyramidale of the CA1 region. Hippocampal field potentials were evoked by fimbrial/commissural stimulation and recorded just before corresponding NMDA applications in the middle panel. Horizontal bars above the pen recorder tracings of extracellular DC potential and above the graphs in the bottom panel indicate NMDA iontophoresis (50 nA, 10 seconds). NMDA-induced DC shifts were increased within the first minute after bilateral carotid artery ligation and inhibited during the later phases of ischemia in parallel with depression of the hippocampal field potential. On reperfusion, a partial recovery was observed in DC shifts along with improvement of the evoked activity. Two successive applications obtained before ischemia and around the 9th minute of ischemia are shown. Deflections at the end of the top tracings measure the local Rt. Note that Rt increases during ischemia. The graphs in the bottom panel illustrate potentiation and subsequent depression of the NMDA-induced Rt increase in the 1st and 11th minutes of ischemia.



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  		Figure 3. Graphs illustrate the changes in the amplitude of DC shifts (A) and local Rt increases (B) induced by NMDA as a function of depression of the evoked activity during ischemia and reperfusion. The data were obtained from 10 rats during repeated applications of NMDA in the stratum radiatum or pyramidale of the CA1 region of the hippocampus. Depression of the evoked activity by ischemia was expressed as percent decrease in the peak amplitude of hippocampal field potential evoked by fimbrial/commissural stimulation. The peak amplitude of DC shifts and the maximal increase in local Rt during NMDA applications were compared with the corresponding mean of six preischemic control applications in every rat (control=100%; error bars indicate SEM). Reperfusion was started after complete loss of the evoked activity. Only NMDA responses recorded after a stable recovery in evoked activity was observed are shown for illustrative reasons. NMDA responses were initially potentiated, subsequently depressed during ischemia, and resumed on reperfusion. The data points on A and B do not match exactly because some of the recordings were not simultaneously obtained.


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  		Table 1. Amplitudes of DC Potentials Induced by NMDA or BAY K8644 During Three Levels of Ischemia and Reperfusion

Effects of Ischemia on BAY K8644 Responses
Pressure application of BAY K8644 for 1 to 5 seconds induced extracellular negative DC potential shifts of 3 to 20 mV that were antagonized by nimodipine (Fig 1Up). On ligation of the carotid arteries, BAY K8644-induced DC shifts were increased up to 183% of their preischemic amplitude, although this effect did not reach statistical significance (P=.07, TableUp; Figs 4Down and 5Down). Potentiation was seen within the first 2 minutes of ischemia when depression of the evoked activity was less than 50%. BAY K8644-induced DC shifts were progressively inhibited with further depression of the field response and abolished when the stimulation-evoked activity disappeared (Figs 4Down and 5Down). Increasing the pressure or duration of application failed to restore BAY K8644 activity. Responses to BAY K8644 recovered concurrently with the evoked activity during reperfusion (Fig 5Down). In two experiments the evoked activity partly recovered after its total failure and then deteriorated again during ischemia. In each instance, BAY K8644 responses simultaneously recovered and deteriorated with the evoked potential, indicating a close association between the two activities. Longer applications (>10 seconds) were required to overcome the local Rt drop caused by pressure application and display the Rt increase induced by BAY K8644. Since recovery took 3 to 5 minutes after long applications, they were not suitable for repeated testing. Therefore, monitoring BAY K8644 action was confined to DC potential changes.



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  		Figure 4. A representative experiment showing the effects of ischemia on BAY K8644-induced DC shifts (bottom tracings). Recordings were done in the stratum radiatum of the hippocampal CA1 region. Top tracings are the hippocampal field potentials evoked by fimbrial/commissural stimulation and were obtained just before BAY K8644 applications. Dots above the bottom tracings indicate BAY K8644 application of 5 psi for 3 seconds. BAY K8644 response was increased within the first minute of bilateral carotid artery ligation and abolished on disappearance of the evoked activity in the 7th minute of ischemia. Deflections at the end of top tracings measure the local Rt. Note the increase in Rt during ischemia.



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  		Figure 5. Graph illustrates the changes in the amplitude of BAY K8644-induced DC shifts as a function of depression of the evoked activity during ischemia. The data were obtained from seven rats during repeated applications of BAY K8644 in the stratum radiatum or pyramidale of the hippocampus. Depression of the evoked activity by ischemia was expressed as percent decrease in the peak amplitude of hippocampal field potential evoked by fimbrial/commissural stimulation. The peak amplitudes of DC shifts were compared with the corresponding mean of six preischemic control applications in every rat (control=100%; error bars indicate SEM). Reperfusion was started after complete loss of the evoked activity. Only BAY K8644 responses recorded after resumption of a stable evoked potential are shown for illustrative reasons. BAY K8644-induced population Ca2+ currents were initially potentiated and subsequently depressed during ischemia.

Intracellular Recordings
CA1 neurons were impaled to assess the direction of RMP alterations during ischemia because both currents studied are voltage dependent and can be inactivated by hyperpolarization or excessive depolarization.23 24 25 26 27 Most of these small neurons were readily depolarized by electrode vibrations due to brain pulsation and exhibited RMPs lower than actual (-38±2 mV, n=30, Fig 6Down), as reported previously.28 To avoid an experimental bias tending to overestimate the mean membrane potential difference between the control and ischemic neurons, we evaluated all penetrations indicating a good electrode sealing even though they had a low membrane potential. On induction of ischemia, neurons further depolarized within a few minutes (-26±3 mV, n=13, P<.001 by Student's t test, Fig 6Down). We were able to obtain stable recordings from two neurons for more than 40 minutes and ligated the carotid arteries while the electrode was still in the cell. RMPs of these cells were -52 and -50 mV and remained steady for 5 minutes before ischemia was induced. After ligation of carotids, they were depolarized by 16 mV in 2 and 5 minutes, respectively, and later by another 10 mV with a slower time course29 30 (Fig 6Down). RMPs of these neurons recovered to -42 and -40 mV after recirculation. Despite limitations in determining the actual RMP, the recorded RMP changes did not support the possibility that the inactivation of NMDA- or BAY K 8644-induced currents was due to hyperpolarization or excessive depolarization.



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  		Figure 6. Resting membrane potentials (A) and intracellular recordings (B) of CA1 neurons recorded before and during ischemia. A, Control recordings were obtained from 30 neurons. To avoid an experimental bias tending to overestimate the mean membrane potential difference between the control and ischemic neurons, we evaluated all penetrations indicating a good electrode sealing even though they had a low membrane potential. During ischemia, all neurons impaled were depolarized, and it was difficult to differentiate between a poor impalement and a satisfactory impalement of a depolarized neuron. Good impalements were determined by considering the membrane input resistance, postsynaptic potentials, and action potentials generated with depolarizing pulses after hyperpolarizing the membrane by current injection. Thirteen neurons depicted on the right, which were picked up by the above criteria, had significantly lower RMPs (P<.001). These neurons were impaled between the 3rd and 20th minutes of ischemia before or after the disappearance of the evoked activity. B, Intracellular recordings from a CA1 neuron obtained with a 3-mol/L KCl electrode (note depolarizing inhibitory postsynaptic potentials). RMPs are indicated on the left of the tracings and time (minutes) after the beginning of ischemia or reperfusion on the right. When the postsynaptic potentials disappeared in the 11th minute of ischemia, hyperpolarizing current injection did not bring back the evoked activity (not shown). Postsynaptic potentials of this cell did not recover during reperfusion, contrary to partial recovery of the CA1 evoked activity.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
Total blockade of NMDA-induced DC shifts and cellular swelling (extracellular resistance increase) by MK-801 strongly suggest that these changes are generated by cation and accompanying water influx through an NMDA receptor ion channel.20 21 22 The changes observed during ischemia in these parameters have therefore been regarded as a measure of altered NMDA receptor activity, although this is an indirect approach and these parameters might be influenced by other factors not related to channel activity. For example, ischemic neuronal swelling might obscure NMDA-induced neuronal swelling if neurons were already maximally swollen. However, we observed parallel changes in NMDA-induced DC shifts and resistance increases not only during mild ischemia when there was no significant cellular swelling but also with deepening of ischemia (compare Fig 3AUp and 3BUp), suggesting that altered NMDA receptor activity was a major determinant of the observed changes. One other factor that might influence channel activities is the shrinkage of extracellular space during ischemia, which may lead to an increase in local drug concentration. However, resistance measurements showed that extracellular space was not shrunken when NMDA effects were potentiated; conversely, it was shrunken when the drug effects were depressed.

The dual effect of in vivo ischemia on NMDA effects is consistent with in vitro findings in hippocampal slices reporting that anoxia can depress or facilitate NMDA currents, depending on experimental conditions.31 32 33 These data also agree with our previous report showing that the NMDA receptor antagonist MK-801 restores partly deteriorated but not severely depressed hippocampal evoked activity during ischemia18 and suggest that one of the reasons for inefficiency of NMDA antagonists during profound ischemia may be the inactivation of NMDA currents in severe energy failure. It is likely that, while increased extracellular concentrations of excitatory amino acids and depolarization of neurons provide a favorable milieu for NMDA receptor activation,34 35 36 37 38 extracellular acidosis, increased Zn2+, {gamma}-aminobutyric acid and adenosine levels, depletion of intracellular energy stores, and rise in intracellular Ca2+ concentration may limit NMDA receptor activation during ischemia.10 39 40 41 42 43 The recent demonstration that a particular redox form of nitric oxide, nitrosonium ion (NO+), can inhibit NMDA receptor activity through interaction with thiol groups44 also suggests that the increase in nitric oxide level in ischemia45 constitutes another, yet less characterized mechanism for the limitation of NMDA receptor activation. Loss of the response to exogenously administered NMDA is unlikely to be due to saturation of NMDA receptors by massive release of glutamate during ischemia because inhibition of the NMDA responses was also observed before ischemia-induced DC shifts developed. It has been shown that substantial rises in extracellular glutamate are consistently accompanied by large DC shifts.46 Moreover, microdialysis studies report a slower rise in extracellular glutamate compared with the rapid decay of NMDA responses in our model. Also, there is currently no evidence to support the unlikely possibility that ischemia alters the sensitivity of NMDA or BAY K8644 to their binding sites.

Our data also indicate that NMDA receptors are functional in ischemia that is not severe enough to cause transmission failure. This finding suggests that NMDA receptor-mediated neurotoxicity must be largely triggered before ischemia deepens and is therefore more likely to prevail in mildly ischemic areas.1 10 47 Studies showing that the NMDA receptor-mediated neurotoxicity is more significant in focal rather than global ischemia and in the penumbra rather than the core ischemic region support this idea.7 8 9 10 Reperfusion also appears to restore favorable conditions for NMDA receptor-mediated neurotoxicity.48

BAY K8644-induced DC shifts are likely to be caused by massive Ca2+ influx through DHP-sensitive Ca2+ channels activated by this powerful agonist. The fact that three Na+ ions are carried into the neurons for extrusion of every Ca2+ ion by Na+-Ca2+ exchanger is very likely to contribute to the development of extracellular negativity. Depolarization induced by cation influx may further facilitate opening of Ca2+ channels and hence may trigger a regenerative cycle. Glial uptake of increased extracellular K+ coming out through Ca2+-activated K+ channels and glutamate release induced by Ca2+ influx may also contribute to DC shifts. Although the ionic basis of BAY K8644-induced extracellular potential shift is not identified, its inhibition by nimodipine suggests that the primary event causing the potential shift is Ca2+ influx through DHP-sensitive Ca2+ channels.

The effect of ischemia on BAY K8644 responses was similar to its effect on NMDA responses. BAY K8644-induced extracellular potential shifts were increased during mild ischemia and depressed with deepening of ischemia. Although potentiation of these DC shifts did not reach statistical significance, possibly because of small sample size, we believe that this effect might be caused by facilitation of voltage-dependent Ca2+ channel activity by ischemic depolarization. On the other hand, inhibition of these responses could be attributed to increased intracellular Ca2+ or failure of channel phosphorylation.42 43 Inhibition of Ca2+ currents has also been demonstrated in hypoglycemia and anoxia in hippocampal slices.49 50 51 These observations were further elaborated by voltage clamping of hippocampal neurons or by recording presynaptic calcium influxes.52 53

In conclusion, these data suggest that NMDA and DHP-sensitive Ca2+ currents are inactivated during ischemia that is severe enough to cause transmission failure. During less intense ischemia and reperfusion, both channels are functional and can be overactivated to cause neurotoxicity.


*    Selected Abbreviations and Acronyms
 
DHP = dihydropyridine
NMDA = N-methyl-D-aspartate
RMP = resting membrane potential
Rt = tissue resistance

Received April 25, 1995; revision received September 12, 1995; accepted October 12, 1995.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

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