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Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo
Pergamon PII:
Neuroscience Vol. 90, No. 3, pp. 771–780, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00493-X
CHANGES IN MEMBRANE PROPERTIES OF CA1 PYRAMIDAL NEURONS AFTER TRANSIENT FOREBRAIN ISCHEMIA IN VIVO T. M. GAO,* W. A. PULSINELLI† and Z. C. XU†‡ *Department of Physiology, The First Military Medical University, Guangzhou, P. R. China †Department of Neurology, University of Tennessee, Memphis, TN 38163, U.S.A.
Abstract—We have previously identified three distinct populations of CA1 pyramidal neurons after reperfusion based on differences in synaptic response, and named these late depolarizing postsynaptic potential neurons (enhanced synaptic transmission), non-late depolarizing postsynaptic potential and small excitatory postsynaptic neurons (depressed synaptic transmission). In the present study, spontaneous activity and membrane properties of CA1 neurons were examined up to 48 h following ⬃ 14 min ischemic depolarization using intracellular recording and staining techniques in vivo. In comparison with preischemic properties, the spontaneous firing rate and the spontaneous synaptic activity of CA1 neurons decreased significantly during reperfusion; spontaneous synaptic activity ceased completely 36–48 h after reperfusion, except for a low level of activity which persisted in non-late depolarizing postsynaptic potential neurons. Neuronal hyperactivity as indicated by increasing firing rate was never observed in the present study. The membrane input resistance and time constant decreased significantly in late depolarizing postsynaptic potential neurons at 24–48 h reperfusion. In contrast, similar changes were not observed in non-late depolarizing postsynaptic potential neurons. The rheobase, spike threshold and spike frequency adaptation in late depolarizing postsynaptic potential neurons increased progressively following reperfusion. Only a transient increase in rheobase and spike threshold was detected in non-late depolarizing postsynaptic potential neurons and spike frequency adaptation remained unchanged in these neurons. The amplitude of fast afterhyperpolarization increased in all neurons after reperfusion, with the smallest increment in non-late depolarizing postsynaptic potential neurons. Small excitatory postsynaptic potential neurons shared similar changes to those of late depolarizing postsynaptic potential neurons. These results suggest that the enhancement and depression of synaptic transmission following ischemia are probably due to changes in synaptic efficacy rather than changes in intrinsic membrane properties. The neurons with enhanced synaptic transmission following ischemia are probably the degenerating neurons, while the neurons with depressed synaptic transmission may survive the ischemic insult. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: ischemia, hippocampus, electrophysiology, in vivo intracellular recording.
CA1 pyramidal neurons in the hippocampus die two to three days after transient cerebral ischemia. 14,19 The mechanisms of this delayed cell death are not clear. It has been hypothesized that neuronal hyperactivity due to an elevation of extracellular excitatory amino acids during ischemia leads to a massive increase in intracellular free Ca 2⫹, which triggers the process of neuronal degeneration. 4,22 However, the dramatically increased extracellular concentration of glutamate and aspartate returns to normal within 30 min after reperfusion. 1,17 This short-lived elevation of excitatory amino acids and its related influx of Ca 2⫹ are insufficient to account for the slow course of cell death in these neurons. Moreover, ‡ To whom correspondence should be addressed at his present address: Department of Anatomy, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, U.S.A.. Abbreviations: EPSP, excitatory postsynaptic potential; fAHP, fast afterhyperpolarization; ISI, interspike interval; L-PSP, late depolarizing postsynaptic potential; PSP, postsynaptic potential; S-EPSP, small excitatory postsynaptic potential.
electrophysiological studies yielded conflicting results regarding neuronal activity in the hippocampus following ischemia. Using extracellular recording techniques in vivo, some investigators reported an increased, 3,24 while others showed a decreased, 2,6,12 firing rate in the CA1 region following ischemia. In a previous study using in vivo intracellular recordings, we reported that spontaneous neuronal activity, evoked postsynaptic potentials and the excitability of CA1 pyramidal neurons were transiently suppressed following 5-min forebrain ischemia; hyperactivity, as indicated by an increase in neuronal firing rate, was not observed up to 7 h after reperfusion. 27 Because 5-min ischemia in our model is insufficient to cause CA1 cell damage, further studies were carried out in animals subjected to an ischemic interval that caused 90% of CA1 cells to die. After such an ischemic insult, a prolonged period of synaptic facilitation was observed in ⬃60% of the CA1 neurons and a prolonged period of synaptic depression was found in ⬃30% of the
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uniform blood glucose levels. For surgical preparation, the animals were anesthetized with 1–2% halothane mixed with 33% O2/66% N2. The arterial po2 was maintained at ⬃120 mmHg and the pco2 at below 40 mmHg. An occluding device (a loop of silicone tubing) was placed loosely around each carotid artery to allow subsequent occlusion of these vessels with minimal mechanical disturbances to the animal. A caudal artery was cannulated for arterial blood pressure monitoring and sampling for blood gas measurements. The animal was then placed on a stereotaxic frame and the core body temperature was maintained at 37⬚C through a temperature control unit (TC-120, Medical System). The vertebral arteries were electrocauterized. A small temperature probe (0.8 mm o.d.) was inserted beneath the skull in the extradural space, after which brain temperature was maintained at 37⬚C with a heating lamp. Severe forebrain ischemia was produced by occluding both common carotid arteries to induce ischemic depolarization for ⬃14 min. Recording of ischemic depolarization
Fig. 1. Evoked postsynaptic responses from CA1 neurons before and after transient forebrain ischemia. (A) Before ischemia, stimulation of the contralateral commissural pathway (CC) elicited an EPSP followed by an inhibitory postsynaptic potential (IPSP). An action potential was triggered from the EPSP. (B) After ischemia, the same stimuli evoked an L-PSP following the initial EPSP from ⬃60% of the CA1 neurons. (C) In ⬃30% of the neurons, contralateral commissural pathway stimuli elicited an EPSP/IPSP with smaller amplitude in comparison with the control neurons. (D) Beginning 24 h after reperfusion, a small EPSP was evoked from ⬃10% of the neurons, even with maximal stimulus intensity. Traces in B–D were recorded 28, 32 and 30 h after reperfusion, respectively. Each trace is the average of four recordings. The action potential in A is truncated. The scales in A apply to B–D.
neurons, suggesting that complex changes of synaptic transmission may be involved in the pathogenesis of neuronal death following ischemia. 7,8 In the present study, we examine the spontaneous activity and membrane properties of CA1 neurons at different periods after transient severe forebrain ischemia using intracellular recording and staining techniques in vivo. Our goal was to characterize the temporal profile of changes in membrane properties of CA1 neurons following severe ischemia and to determine whether such events could explain the changes in synaptic transmission after ischemia. Preliminary results have been presented in abstract form. EXPERIMENTAL PROCEDURES
Transient forebrain ischemia Male adult Wistar rats (250–350 g; Sasco) were used in the present study. The NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) was strictly followed. Transient forebrain ischemia was induced using the four-vessel occlusion method, 18 with some modifications. The animals were fasted overnight to provide
The extracellular d.c. potential in the CA1 region was monitored during ischemia. Recording microelectrodes were pulled from glass capillaries with filaments (A-M system) to a tip resistance of ⬃5 MV when filled with 2 M NaCl. A burr hole was drilled at 4.0 mm anterior to the interaural line and 3.0 mm from the midline. A microelectrode was inserted into the CA1 region of the hippocampus (⬃2.5 mm below the brain surface). Following the baseline recording, forebrain ischemia was initiated by tightening the occluding device. Ischemic depolarization as indicated by d.c. potential shifting from 0 to ⫺20 mV occurs approximately 2.5 min after occlusion. Occlusion of carotid arteries was terminated at 12 min after the onset of ischemic depolarization. Upon releasing the common carotid arteries, cerebral blood flow resumed immediately and the d.c. potential gradually returned to 0 mV in approximately 2 min. The duration of ischemic depolarization was measured from the onset of negative deflection of the d.c. potential to the point at which the d.c. potential completely returned to preischemic levels after recirculation. Only animals with ischemic depolarizations of ⬃14 min were used for electrophysiological experiments. For recordings longer than 12 h after reperfusion, animals were returned to the cage after reperfusion and allowed free access to water and food. The animals were then re-anesthetized and prepared for recording at different time-points after reperfusion. Intracellular recording and staining Preparation for intracellular recording and staining in vivo was performed as described previously. 26,27 In brief, under 1–2% halothane anesthesia, the skull was opened to expose the recording site and for placement of stimulus electrodes. Recording electrodes were pulled from glass capillaries with filaments to a tip resistance of 50–80 MV when filled with a solution of 3% neurobiotin (Vector) in 2 M potassium acetate. Cerebrospinal fluid was drained via a cisternal puncture to reduce brain pulsation and the animal was suspended by a clamp applied to the tail. After placement of a microelectrode in the cortex above the hippocampus for recording, the exposed surface of the brain was covered with soft paraffin wax. The microelectrode was advanced slowly into the hippocampus to impale CA1 neurons. After impalement, the neurons with a stable membrane potential of ⫺60 mV or greater were selected for further study. Data were digitized and stored on a Macintosh computer using the data acquisition program Axodata (Axon Instruments). The Student’s t-test or ANOVA was used for statistical analysis (Staview, Abacus Concepts). After each successful recording, neurobiotin was
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Fig. 2. Spontaneous activity of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. Before ischemia, the CA1 neuron exhibited active spontaneous membrane potential fluctuation. The action potential in this trace is truncated. After ischemia, spontaneous firing of all neurons disappeared. The spontaneous synaptic activity in L-PSP neurons decreased significantly by 3 h and ceased by 41 h following reperfusion. However, the non-L-PSP neurons still maintained spontaneous synaptic activity at this time. No spontaneous synaptic activity was observed in S-EPSP neurons. The scales apply to all recordings.
iontophoresed into the cell by passing depolarizing current pulses (2 Hz, 300 ms, 0.5–1 nA) for 10 min. At the end of the experiment, the animal was deeply anesthetized and perfused transcardially with 0.01 M phosphate-buffered saline followed by 4% paraformaldehyde. The brain was removed and stored in fixative overnight. Coronal sections were cut at 50 mm thickness using a Vibratome and incubated in 0.1% horseradish peroxidase-conjugated avidin-D (Vector) in 0.01 M potassium phosphate-buffered saline (pH 7.4) with 0.5% Triton X-100 overnight at room temperature. After detection of peroxidase activity with 3,3 0 -diaminobenzidine, sections were examined in potassium phosphatebuffered saline. Sections containing labeled neurons were mounted on gelatin-coated slides and counterstained with Cresyl Violet for light microscopy.
RESULTS
Experiments were performed on control rats (n 10) and rats subjected to forebrain ischemia with ischemic depolarization of 13.8 ^ 0.9 min (n 37). This degree and duration of ischemia consistently produced selective cell death in the CA1 region of rat hippocampus using the fourvessel occlusion method under halothane anesthesia. 21 The arterial gas tension (pco2, po2, pH, glucose and hemoglobin) and general physiological condition (arterial blood pressure, rectal and brain temperature) of the animals before and after ischemia did not differ and were maintained within
normal ranges throughout the experiments. A total of 128 neurons was analysed in the present study, of which 110 were successfully stained and identified as CA1 pyramidal cells. The 18 unidentified cells, considered as CA1 pyramidal neurons based on stereotaxic parameters and electrophysiological characteristics, were also included in the present study. The control data presented in this report were derived from 22 neurons recorded before ischemia. Intracellular recording of CA1 neurons was very difficult two days after ischemia, especially in the medial portion of the CA1 zone. Among 10 animals in which recordings were attempted during 37–48 h after reperfusion, five showed unequivocal cell degeneration in the medial protion of the CA1 zone with hematoxylin–eosin staining. However, no gross signs of degeneration, such as swelling, shrinkage of cell body or fragmentation of dendrites, were observed in postischemic neurons having undergone intracellular recording and labeled with neurobiotin. In a previous report, 7 we identified three distinct populations of CA1 pyramidal neurons after reperfusion based on difference in synaptic response, and named these late depolarizing postsynaptic potential (L-PSP), non-L-PSP and small excitatory postsynaptic potential (S-EPSP) neurons (Fig. 1). In order to study the temporal changes of membrane properties following reperfusion, the
⫺73 ⫺73 ⫺70 ⫺70 ⫺69
^ 6.28 (22) ^ 4.75 (11) ^ 5.41 (22) ^ 6.21 (18) ^ 4.82 (12)
RMP (mV) 77 81 86 79 79
^ 7.52 (22) ^ 12.09 (10) ^ 10.47** (20) ^ 9.70 (13) ^ 7.73 (9)
Spike height (mV) 0.82 ^ 0.20 (22) 0.96 ^ 0.24 (10) 0.85 ^ 0.15 (20) 1.07 ^ 0.51* (13) 0.74 ^ 0.16 (9)
Spike width (ms) 25.63 ^ 5.51 (15) 22.04 ^ 9.25 (7) 22.02 ^ 6.22 (16) 14.80 ^ 8.84** (9) 16.68 ^ 7.20** (9)
Rin (MV) 12.44 ^ 2.85 (15) 9.87 ^ 2.97 (7) 12.53 ^ 4.99 (16) 6.98 ^ 3.63** (9) 7.94 ^ 2.52** (9)
Time constant (ms)
0.53 ^ 0.58 (16) 0.00 ^ 0.00** (11) 0.02 ^ 0.11** (22) 0.00 ^ 0.01** (18) 0.00 ^ 0.00** (12)
Spont. firing (Hz)
77 ^ 7.52 (22) 77 ^ 10.14 (11) 74 ^ 8.34 (3) 84 ^ 7.00 (7) 78 ^ 11.87 (6)
⫺73 ^ 6.28 (22) ⫺69 ^ 7.06 (13) ⫺67 ^ 3.17 (4) ⫺69 ^ 4.12 (7) ⫺69 ^ 2.38 (7)
Before ⬃ 5 h after ⬃ 19 h after ⬃ 31 h after ⬃ 42 h after
0.82 ^ 0.20 (22) 0.84 ^ 0.11 (11) 0.76 ^ 0.16 (3) 0.89 ^ 0.12 (7) 0.69 ^ 0.09 (6)
Spike width (ms) 25.63 ^ 5.51 (15) 22.94 ^ 7.68 (12) 24.23 ^ 5.93 (3) 28.68 ^ 3.45 (5) 20.25 ^ 3.90 (4)
Rin (MV)
12.44 ^ 2.88 (15) 14.25 ^ 6.03 (12) 13.50 ^ 3.64 (3) 10.96 ^ 2.04 (5) 7.98 ^ 0.53 (4)
Time constant (ms)
0.53 ^ 0.58 (16) 0.05 ^ 0.14** (13) 0.03 ^ 0.07** (4) 0.03 ^ 0.07** (7) 0.00 ^ 0.03** (7)
Spont. firing (Hz)
77 ^ 7.52 (22) 78 ^ 12.11 (5) 77 ^ 16.26 (3)
⫺73 ^ 6.28 (22) ⫺71 ^ 7.21 (7) ⫺69 ^ 5.32 (4)
Before ⬃ 31 h after ⬃ 42 h after
0.82 ^ 0.20 (22) 0.85 ^ 0.20 (5) 0.75 ^ 0.21 (3)
Spike width (ms)
25.63 ^ 5.51 (15) 15.73 ^ 11.34* (4) 12.50 ^ 10.54* (3)
Rin (MV)
12.44 ^ 2.88 (15) 7.35 ^ 3.49** (4) 6.57 ^ 6.03** (3)
Time constant (ms)
0.53 ^ 0.58 (16) 0.00 ^ 0.00** (7) 0.01 ^ 0.03** (4)
Spont. firing (Hz)
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. *P ⬍ 0.05, **P ⬍ 0.01, ANOVA.
Spike height (mV)
RMP (mV)
Reperfusion
Table 3. Membrane properties of small excitatory postsynaptic potential neurons at different periods after ischemia
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. **P ⬍ 0.01, ANOVA.
Spike height (mV)
RMP (mV)
Reperfusion
Table 2. Membrane properties of non-late depolarizing postsynaptic potential neurons at different periods after ischemia
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. *P ⬍ 0.05, **P ⬍ 0.01, ANOVA.
Before ⬃5 h after ⬃19 h after ⬃31 h after ⬃42 h after
Reperfusion
Table 1. Electrophysiological properties of late depolarizing postsynaptic potential neurons at different periods after reperfusion
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Fig. 3. Current–voltage (I–V) relationship of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. Each trace is the average of four recordings. Top panels are membrane potential deflections caused by the current pulses. Bottom panels are intracellularly applied current pulses. (A) I–V relationship before ischemia. A marked anomalous rectification was observed in this neuron. (B–D) I–V relationship of representative L-PSP, non-L-PSP and S-EPSP neurons, respectively. Scales in D apply to A–C. The action potentials in these figures are truncated. (E–G) Plots of I–V curve from control and three groups of neurons, showing the input resistance changes at different intervals after reperfusion. Beginning on the second day following reperfusion, the slope of the I–V curve of L-PSP neurons (E) and S-EPSP neurons (G) was persistently smaller than that of controls, indicating the reduction in input resistance. At the same time, the inward rectification with depolarizing currents changed to an outward rectification in these two groups of neurons and this outward rectification increased progressively after reperfusion. The I–V curves of non-L-PSP neurons at different intervals after reperfusion were about the same as before ischemia (F).
neurons within these three groups were pooled according to the reperfusion time at which they were recorded: 0–12 h (median: 5 h); 12–24 h (median: 19 h); 24– 36 h (median: 31 h); 36–48 h (median: 42 h). Spontaneous activity after reperfusion Before ischemia, the spontaneous firing rate of CA1 pyramidal neurons was 0.53 ^ 0.58 spikes/s (n 16). Spontaneous neuronal firing ceased shortly after ischemia in all three groups of neurons and remained markedly suppressed up to 48 h following reperfusion (Tables 1–3). Neuronal hyperactivity as
indicated by increased spontaneous firing rate was never observed in the present study. Spontaneous synaptic activity as indicated by small PSPs superimposed on baseline membrane potentials was very active in CA1 neurons before ischemia. Spontaneous synaptic activity in L-PSP and non-L-PSP neurons decreased significantly 3 h after reperfusion and was completely abolished 36–48 h after reperfusion in L-PSP neurons (Fig. 2). Spontaneous synaptic activity in non-L-PSP neurons, however, remained at low levels 36–48 h after reperfusion. No spontaneous synaptic activities were observed in S-EPSP neurons.
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passive membrane conductance, the active membrane conductance of CA1 neurons also changed remarkably after ischemia. As shown in Fig. 3, CA1 neurons before ischemia exhibited an obvious inward rectification when membrane potential was maintained at depolarizing or hyperpolarizing extremes by current injection. The latter results are similar to previous observations. 11,20 After ischemia, the inward rectification with depolarizing currents changed to an outward rectification in both L-PSP and S-EPSP neurons, and this outward rectification increased progressively following reperfusion (Fig. 3E, G). No such change was observed in non-L-PSP neurons (Fig. 3F). The alteration of inward rectification with hyperpolarizing currents in all three groups of neurons was not as great as that in the depolarizing extreme (Fig. 3E–G). The time constant of CA1 neurons after reperfusion showed similar changes as those of input resistance. The time constant of L-PSP and S-EPSP neurons decreased to approximately 50% of preischemic values 31–42 h after reperfusion (P ⬍ 0.01, ANOVA; Tables 1 and 3). In non-L-PSP neurons, the time constant remained normal until after ⬃31 h and then declined at 42 h following reperfusion (Table 2). Excitability, repetitive firing and fast afterhyperpolarization after reperfusion Fig. 4. Plots showing the changes in spike threshold (A) and rheobase (B) of CA1 pyramidal neurons at different intervals after reperfusion. The value of each plot is the mean, with the number of neurons in parentheses. The spike threshold and rheobase of L-PSP and S-EPSP neurons increased gradually following reperfusion, indicating a progressive decrease in neuronal excitability. However, a transient increase in spike threshold and rheobase was observed in non-L-PSP neurons at the early reperfusion period.
Input resistance and time constant after reperfusion To compare the current–voltage relationship of CA1 neurons before and after ischemia, constantcurrent pulses (200 ms, ⫺1.0 to ⫹0.5 nA) were passed; the voltage values were measured from the averages of four recordings at the steady state of the transients (average between 160 and 180 ms from the beginning of the pulse). The membrane input resistance of CA1 neurons was 25.63 ^ 5.51 MV (n 15) in control animals. The input resistance of L-PSP neurons remained unchanged during the first 24 h after reperfusion, but decreased significantly to 14.80 and 16.68 MV at 31 and 42 h after reperfusion, respectively (P ⬍ 0.01, ANOVA; Table 1). The input resistance of non-L-PSP neurons, in contrast, displayed no significant changes up to 48 h following reperfusion (Table 2). The input resistance of S-EPSP neurons was decreased significantly, to 15.73 and 12.50 MV at 31 and 42 h after reperfusion, respectively (P ⬍ 0.05, ANOVA; Table 3). In addition to the dramatic alterations in
The changes in neuronal excitability were evaluated by comparing the spike threshold and rheobase of CA1 neurons before and after ischemia. The spike threshold was measured at the beginning of the upstroke of the first action potential and the rheobase was determined as the minimal intensity of depolarizing current pulse to trigger an action potential. As shown in Fig. 4, the spike threshold and rheobase of L-PSP and S-EPSP neurons increased progressively after reperfusion, while only a transient increase in spike threshold (P ⬍ 0.05, ANOVA) and rheobase (P ⬍ 0.01, ANOVA) was detected in non-L-PSP neurons at 5 h after reperfusion. Repetitive firing was induced by application of depolarizing current pulses (400 ms, 0.1–1.6 nA). In control neurons, there was a strong spike frequency adaptation of spike trains during application of constant-current pulses, as indicated by the differences in spike frequency between the first interspike interval (ISI) and the second ISI (Fig. 5). The repetitive firing of L-PSP neurons was dramatically depressed after ischemic insult. The spike frequency declined progressively at all current intensities, with the lowest firing rate at 42 h after reperfusion (Fig. 5D). In contrast, the spike frequency of non-L-PSP neurons remained about the same as that of controls, except the frequency of the second ISI increased at stronger depolarizing current pulses following reperfusion (Fig. 5E). In CA1 pyramidal neurons, each action potential is followed by a fast afterhyperpolarization (fAHP),
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Fig. 5. Repetitive firing of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. (A–C) Spike trains were evoked by 0.6-nA depolarizing current pulses. Examples of repetitive firing of a control neuron (A), an L-PSP neuron 34 h after reperfusion (B) and a non-L-PSP neuron 32 h after reperfusion (C). Scales in B apply to A and C. (D, E) Plots showing the changes in the relationship between depolarizing current and spike frequency of the first ISI (upper panel) and second ISI (lower panel) of L-PSP and non-L-PSP neurons at different intervals after reperfusion. Each point is the mean value. The spike frequency increased linearly with the increase of current intensities. In comparison with control values, the spike frequency of both the first and second ISIs decreased progressively in L-PSP neurons following reperfusion, indicating a gradual suppression of repetitive firing. However, in non-L-PSP neurons, the spike frequency of the first ISI remained unchanged after reperfusion, although there was an increase in the spike frequency of the second ISI at high current intensities.
which is mediated by calcium-activated potassium current. 15,23 In the present study, the fAHP was induced by the injection of a depolarizing current pulse that generates action potentials. The amplitude of the fAHP was measured as deviation from the beginning of the upstroke of an action potential within 5 ms after the peak of a single spike. The amplitude of the fAHP increased significantly, from around 1 mV in control neurons to approximately 5 mV in L-PSP neurons (P ⬍ 0.01, ANOVA). The amplitude of the fAHP in L-PSP neurons at different intervals following reperfusion was slightly different, with the highest value at ⬃ 5 h and the lowest value at ⬃ 31 h after reperfusion (Fig. 6E). Although the amplitude of the fAHP in non-L-PSP neurons also increased after reperfusion and showed a similar pattern to that of L-PSP neurons, the amplitude of the fAHP in non-L-PSP neurons was approximately 2 mV smaller than that of L-PSP neurons at each reperfusion interval (Fig. 6E). The amplitude of the fAHP in S-EPSP neurons was the same as that in L-PSP neurons. DISCUSSION
The present study demonstrates that the postischemic membrane properties differ among the three groups of CA1 neurons distinguished by their
postischemic synaptic transmission properties (i.e. L-PSP, non-L-PSP and S-EPSP neurons). 7 The differences in synaptic transmission and membrane properties of these postischemic neurons suggest that they may have different histopathological outcomes. Alteration of input resistance following reperfusion In agreement with previous studies, 13 we observed that the membrane input resistance of CA1 neurons within 24 h reperfusion remained similar to control neurons. However, after 24 h reperfusion, the input resistance declined significantly in LPSP and S-EPSP neurons, but did not change in nonL-PSP neurons. The decrease of input resistance in L-PSP and S-EPSP neurons 24–48 h after reperfusion differs from the results of previous studies. It has been reported that no difference was found in the input resistance of CA1 neurons between control hippocampal slices and slices prepared 24 or 48 h after 20-min ischemia. 10,13 It is difficult to reconcile that membrane input resistance of CA1 neurons 24– 48 h after reperfusion remains normal, while morphological signs of cell degeneration were already apparent. 10 The reduced input resistance observed in L-PSP and S-EPSP neurons in the present study may be due to the loss of functional membrane integrity and disruption of ion conductance
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associated with pathological changes after ischemia. The unchanged input resistance of non-L-PSP neurons, however, suggests that these neurons may survive the ischemic insult or undergo degenerating pathways different from those of L-PSP and S-EPSP neurons. Depression of neuronal excitability following reperfusion
Fig. 6. Fast afterhyperpolarization (fAHP) of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. fAHPs were induced by action potentials evoked by a depolarizing current pulse. (A–D) The amplitude of the fAHP increased in all three groups of postischemic neurons. Scales in D apply to A–C. The action potentials in these figures are truncated. (E) Plots showing the changes in amplitude of the fAHP in CA1 neurons after reperfusion. Each point is the mean value. In L-PSP and S-EPSP neurons, the amplitude of the fAHP was persistently larger than that of control neurons (P ⬍ 0.01, ANOVA) during the whole course of reperfusion. Although the non-L-PSP neurons showed similar changes to those of LPSP neurons, the amplitude of the fAHP in these neurons was obviously lower than those in L-PSP and S-EPSP neurons at each corresponding reperfusion time.
The gradual increase in the spike threshold in LPSP and S-EPSP neurons indicates that neuronal excitability in these cells is progressively depressed following reperfusion. In contrast, only a transient increase in spike threshold was observed in non-LPSP neurons. Using the gerbil brain slice preparation at different time-points after ischemia, Urban et al. 25 reported that the excitability of CA1 neurons was depressed in a similar time-dependent fashion. The depression of neuronal excitability may be responsible for the decrease in spontaneous firing rate of CA1 neurons following ischemia, as observed in the present study and in previous reports. 2,6,12,27 Increased spike threshold after reperfusion accounts for many postischemic changes observed in the present study. For example, the increased rheobase after reperfusion is closely related to the increase in spike threshold and it may also contribute to the depression of repetitive firing after reperfusion. Increased spike threshold after reperfusion has also been observed in spiny neurons in the neostriatum. 26 The mechanism underlying increased spike threshold after reperfusion in neostriatal neurons may reflect dopamine effects on D1 dopamine receptors. 26 It has been shown that decreased excitability of CA1 neurons during anoxia is due to the increase in potassium conductance. 5,9,16 The mechanisms causing the increase of spike threshold in CA1 neurons after reperfusion are not clear at this point. Two different types of changes in neuronal excitability were observed in CA1 neurons after reperfusion: a progressive increase in spike threshold was detected in L-PSP and S-EPSP neurons, while a transient increase in spike threshold was observed in non-L-PSP neurons and in CA1 neurons following 5-min ischemia. 27 Since ischemia of 5 min duration will not cause neuronal degeneration in the rat hippocampus, the transient increase in spike threshold is probably a compensatory response to the ischemic stress rather than an event associated with the process of cell death. The progressive increase in spike threshold in L-PSP and S-EPSP neurons, however, may be a sign of membrane degradation following ischemia. The progressive decrease in input resistance in these neurons also supports this notion. CONCLUSIONS
The differences in membrane properties between L-PSP and non-L-PSP neurons following ischemia
Membrane properties of CA1 neurons after ischemia
strongly indicate that these two groups of neurons behave differently to severe ischemia. L-PSP neurons show an enhanced synaptic transmission and a progressive decrease in excitability following reperfusion, while non-L-PSP neurons show a depressed synaptic transmission and transient decrease in excitability following ischemia. During the first 12 h of reperfusion, the synaptic transmission is enhanced in L-PSP neurons and depressed in non-L-PSP neurons. 7 However, the reduced excitability of both groups of neurons with an unchanged input resistance suggests that the postischemic changes in synaptic transmission in CA1 neurons are not due to the alterations in their membrane properties. The late but progressive decrease in
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input resistance in L-PSP and S-EPSP neurons supports the notion that these neurons are the degenerating neurons following severe ischemic insult. In contrast, the transient decrease in excitability and unchanged input resistance following reperfusion in non-L-PSP neurons suggest that they may have different postischemic outcome to that of L-PSP and S-EPSP neurons.
Acknowledgements—This research was supported by grants AHA 94008130, NIH NS33101 to Z.C.X. and SemmesMurphey Chair of Excellence to W.A.P. T.M.G. is the recipient of postdoctoral fellowships from the Neuroscience Center of Excellence in U.T. Memphis and grants NNSF 39570263 and NSFGP 950537 of P. R. China.
REFERENCES
1. Benveniste H., Drejer J., Schousboe A. and Diemer N. (1984) Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369–1374. 2. Buzsa´ki G., Freund T. F., Bayardo F. and Somogyi P. (1989) Ischemia-induced changes in the electrical activity of the hippocampus. Expl Brain Res. 78, 268–278. 3. Chang H. S., Sasaki T. and Kassel N. F. (1989) Hippocampal unit activity after transient ischemia in rats. Stroke 20, 1051– 1058. 4. Choi D. W. and Rothman S. M. (1990) The role of glutamate neurotoxicity in hypoxic–ischemic neuronal death. A. Rev. Neurosci. 13, 171–182. 5. Fujiwara N., Higashi H., Shimoji K. and Yoshimura M. (1987) Effects of hypoxia on rat hippocampal neurons in vitro. J. Physiol. 384, 131–151. 6. Furukawa K., Yamana K. and Kogure K. (1990) Postischemic alterations of spontanous activities in rat hippocampal CA1 neurons. Brain Res. 530, 257–260. 7. Gao T. M., Pulsinelli W. A. and Xu Z. C. (1998) Prolonged enhancement and depression of synaptic transmission in CA1 pyramidal neurons induced by transient forebrain ischemia in vivo. Neuroscience 87, 371–383. 8. Gao T. M. and Xu Z. C. (1996) In vivo intracellular demonstration of an ischemia-induced postsynaptic potential from CA1 pyramidal neurons in rat hippocampus. Neuroscience 75, 665–669. 9. Hansen A. J., Hounsgaard J. and Jahnsen H. (1982) Anoxia increases potassium conductance in hippocampal nerve cells. Acta physiol. scand. 115, 301–310. 10. Hori N. and Carpenter D. O. (1994) Functional and morphological changes induced by transient in vivo ischemia. Expl Neurol. 129, 279–289. 11. Hotson J. R., Prince D. A. and Schwartzkroin P. A. (1983) Anomalous inward rectification in hippocampal neurons. J. Neurophysiol. 42, 889–895. 12. Iomn H., Mitani A., Andou Y., Arai T. and Kataoka K. (1991) Delayed neuronal death is induced without postischemic hyperexcitability: continuous multiple-unit recording from ischemic CA1 neurons. J. cerebr. Blood Flow Metab. 11, 819– 823. 13. Jensen M. S., Lambert J. D. C. and Johansen F. F. (1991) Electrophysiological recordings from rat hippocampus slices following in vivo brain ischemia. Brain Res. 554, 166–175. 14. Kirino T. (1982) Delayed neuronal death in gerbil hippocampus following ischemia. Brain Res. 237, 57–69. 15. Lancaster B. and Nicoll R. A. (1987) Properties of two calcium-activated hyperpolarizations in rat hippocampal neurons. J. Physiol. 389, 187–203. 16. Lebond J. and Krenjevic K. (1989) Hypoxic changes in hippocampal neurons. J. Neurophysiol. 62, 1–14. 17. Mitani A., Andou Y. and Kataoka K. (1992) Selective vulnerability of hippocampal CA 1 neurons cannot be explained in terms of an increase in glutamate concentration during ischemia in gerbil: brain microdialysis study. Neuroscience 48, 307–313. 18. Pulsinelli W. A. and Brierley J. B. (1979) A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke 10, 267–272. 19. Pulsinelli W. A., Brierley J. B. and Plum F. (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11, 491–498. 20. Purpura D. P., Prelevic S. and Santini M. (1968) Hyperpolarizing increase in membrane conductance in hippocampal neurons. Brain Res. 7, 310–312. 21. Ren Y. B. and Xu Z. C. (1995) Halothane effect on ischemic outcome and temperature pattern following transient ischemia in rats. Soc. Neurosci. Abstr. 21, 393.18. 22. Rothman S. M. and Olney J. W. (1986) Glutamate and the pathophysiology of hypoxic–ischemic brain damage. Ann. Neurol. 19, 105–111. 23. Storm J. F. (1987) Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. J. Physiol. 385, 733–759. 24. Suzuki R., Yamaguchi T., Li C. L. and Klatzo I. (1983) The effects of 5-minute ischemia in mongolian gerbils. II. Changes of spontaneous neuronal activity in cerebral cortex and CA1 sector of hippocampus. Acta neuropath., Berlin 60, 217–222. 25. Urban L., Neill K., Crain B., Nadler J. V. and Somjen G. (1989) Postischemic synaptic physiology in area CA1 of the gerbil hippocampus studied in vitro. J. Neurosci. 9, 3966–3975.
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26. Xu Z. C. (1995) Neurophysiological changes of spiny neurons in rat neostriatum after transient forebrain ischemia: an in vivo intracellular recording and staining study. Neuroscience 67, 823–836. 27. Xu Z. C. and Pulsinelli W. A. (1996) Neurophysiological changes of CA1 pyramidal neurons following transient ischemia: an in vivo intracellular recording and staining study. J. Neurophysiol. 76, 1689–1697. (Accepted 21 August 1998)
Neuroscience Vol. 90, No. 3, pp. 771–780, 1999 Copyright 䉷 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 S0306-4522(98)00493-X
CHANGES IN MEMBRANE PROPERTIES OF CA1 PYRAMIDAL NEURONS AFTER TRANSIENT FOREBRAIN ISCHEMIA IN VIVO T. M. GAO,* W. A. PULSINELLI† and Z. C. XU†‡ *Department of Physiology, The First Military Medical University, Guangzhou, P. R. China †Department of Neurology, University of Tennessee, Memphis, TN 38163, U.S.A.
Abstract—We have previously identified three distinct populations of CA1 pyramidal neurons after reperfusion based on differences in synaptic response, and named these late depolarizing postsynaptic potential neurons (enhanced synaptic transmission), non-late depolarizing postsynaptic potential and small excitatory postsynaptic neurons (depressed synaptic transmission). In the present study, spontaneous activity and membrane properties of CA1 neurons were examined up to 48 h following ⬃ 14 min ischemic depolarization using intracellular recording and staining techniques in vivo. In comparison with preischemic properties, the spontaneous firing rate and the spontaneous synaptic activity of CA1 neurons decreased significantly during reperfusion; spontaneous synaptic activity ceased completely 36–48 h after reperfusion, except for a low level of activity which persisted in non-late depolarizing postsynaptic potential neurons. Neuronal hyperactivity as indicated by increasing firing rate was never observed in the present study. The membrane input resistance and time constant decreased significantly in late depolarizing postsynaptic potential neurons at 24–48 h reperfusion. In contrast, similar changes were not observed in non-late depolarizing postsynaptic potential neurons. The rheobase, spike threshold and spike frequency adaptation in late depolarizing postsynaptic potential neurons increased progressively following reperfusion. Only a transient increase in rheobase and spike threshold was detected in non-late depolarizing postsynaptic potential neurons and spike frequency adaptation remained unchanged in these neurons. The amplitude of fast afterhyperpolarization increased in all neurons after reperfusion, with the smallest increment in non-late depolarizing postsynaptic potential neurons. Small excitatory postsynaptic potential neurons shared similar changes to those of late depolarizing postsynaptic potential neurons. These results suggest that the enhancement and depression of synaptic transmission following ischemia are probably due to changes in synaptic efficacy rather than changes in intrinsic membrane properties. The neurons with enhanced synaptic transmission following ischemia are probably the degenerating neurons, while the neurons with depressed synaptic transmission may survive the ischemic insult. 䉷 1999 IBRO. Published by Elsevier Science Ltd. Key words: ischemia, hippocampus, electrophysiology, in vivo intracellular recording.
CA1 pyramidal neurons in the hippocampus die two to three days after transient cerebral ischemia. 14,19 The mechanisms of this delayed cell death are not clear. It has been hypothesized that neuronal hyperactivity due to an elevation of extracellular excitatory amino acids during ischemia leads to a massive increase in intracellular free Ca 2⫹, which triggers the process of neuronal degeneration. 4,22 However, the dramatically increased extracellular concentration of glutamate and aspartate returns to normal within 30 min after reperfusion. 1,17 This short-lived elevation of excitatory amino acids and its related influx of Ca 2⫹ are insufficient to account for the slow course of cell death in these neurons. Moreover, ‡ To whom correspondence should be addressed at his present address: Department of Anatomy, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, U.S.A.. Abbreviations: EPSP, excitatory postsynaptic potential; fAHP, fast afterhyperpolarization; ISI, interspike interval; L-PSP, late depolarizing postsynaptic potential; PSP, postsynaptic potential; S-EPSP, small excitatory postsynaptic potential.
electrophysiological studies yielded conflicting results regarding neuronal activity in the hippocampus following ischemia. Using extracellular recording techniques in vivo, some investigators reported an increased, 3,24 while others showed a decreased, 2,6,12 firing rate in the CA1 region following ischemia. In a previous study using in vivo intracellular recordings, we reported that spontaneous neuronal activity, evoked postsynaptic potentials and the excitability of CA1 pyramidal neurons were transiently suppressed following 5-min forebrain ischemia; hyperactivity, as indicated by an increase in neuronal firing rate, was not observed up to 7 h after reperfusion. 27 Because 5-min ischemia in our model is insufficient to cause CA1 cell damage, further studies were carried out in animals subjected to an ischemic interval that caused 90% of CA1 cells to die. After such an ischemic insult, a prolonged period of synaptic facilitation was observed in ⬃60% of the CA1 neurons and a prolonged period of synaptic depression was found in ⬃30% of the
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uniform blood glucose levels. For surgical preparation, the animals were anesthetized with 1–2% halothane mixed with 33% O2/66% N2. The arterial po2 was maintained at ⬃120 mmHg and the pco2 at below 40 mmHg. An occluding device (a loop of silicone tubing) was placed loosely around each carotid artery to allow subsequent occlusion of these vessels with minimal mechanical disturbances to the animal. A caudal artery was cannulated for arterial blood pressure monitoring and sampling for blood gas measurements. The animal was then placed on a stereotaxic frame and the core body temperature was maintained at 37⬚C through a temperature control unit (TC-120, Medical System). The vertebral arteries were electrocauterized. A small temperature probe (0.8 mm o.d.) was inserted beneath the skull in the extradural space, after which brain temperature was maintained at 37⬚C with a heating lamp. Severe forebrain ischemia was produced by occluding both common carotid arteries to induce ischemic depolarization for ⬃14 min. Recording of ischemic depolarization
Fig. 1. Evoked postsynaptic responses from CA1 neurons before and after transient forebrain ischemia. (A) Before ischemia, stimulation of the contralateral commissural pathway (CC) elicited an EPSP followed by an inhibitory postsynaptic potential (IPSP). An action potential was triggered from the EPSP. (B) After ischemia, the same stimuli evoked an L-PSP following the initial EPSP from ⬃60% of the CA1 neurons. (C) In ⬃30% of the neurons, contralateral commissural pathway stimuli elicited an EPSP/IPSP with smaller amplitude in comparison with the control neurons. (D) Beginning 24 h after reperfusion, a small EPSP was evoked from ⬃10% of the neurons, even with maximal stimulus intensity. Traces in B–D were recorded 28, 32 and 30 h after reperfusion, respectively. Each trace is the average of four recordings. The action potential in A is truncated. The scales in A apply to B–D.
neurons, suggesting that complex changes of synaptic transmission may be involved in the pathogenesis of neuronal death following ischemia. 7,8 In the present study, we examine the spontaneous activity and membrane properties of CA1 neurons at different periods after transient severe forebrain ischemia using intracellular recording and staining techniques in vivo. Our goal was to characterize the temporal profile of changes in membrane properties of CA1 neurons following severe ischemia and to determine whether such events could explain the changes in synaptic transmission after ischemia. Preliminary results have been presented in abstract form. EXPERIMENTAL PROCEDURES
Transient forebrain ischemia Male adult Wistar rats (250–350 g; Sasco) were used in the present study. The NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23) was strictly followed. Transient forebrain ischemia was induced using the four-vessel occlusion method, 18 with some modifications. The animals were fasted overnight to provide
The extracellular d.c. potential in the CA1 region was monitored during ischemia. Recording microelectrodes were pulled from glass capillaries with filaments (A-M system) to a tip resistance of ⬃5 MV when filled with 2 M NaCl. A burr hole was drilled at 4.0 mm anterior to the interaural line and 3.0 mm from the midline. A microelectrode was inserted into the CA1 region of the hippocampus (⬃2.5 mm below the brain surface). Following the baseline recording, forebrain ischemia was initiated by tightening the occluding device. Ischemic depolarization as indicated by d.c. potential shifting from 0 to ⫺20 mV occurs approximately 2.5 min after occlusion. Occlusion of carotid arteries was terminated at 12 min after the onset of ischemic depolarization. Upon releasing the common carotid arteries, cerebral blood flow resumed immediately and the d.c. potential gradually returned to 0 mV in approximately 2 min. The duration of ischemic depolarization was measured from the onset of negative deflection of the d.c. potential to the point at which the d.c. potential completely returned to preischemic levels after recirculation. Only animals with ischemic depolarizations of ⬃14 min were used for electrophysiological experiments. For recordings longer than 12 h after reperfusion, animals were returned to the cage after reperfusion and allowed free access to water and food. The animals were then re-anesthetized and prepared for recording at different time-points after reperfusion. Intracellular recording and staining Preparation for intracellular recording and staining in vivo was performed as described previously. 26,27 In brief, under 1–2% halothane anesthesia, the skull was opened to expose the recording site and for placement of stimulus electrodes. Recording electrodes were pulled from glass capillaries with filaments to a tip resistance of 50–80 MV when filled with a solution of 3% neurobiotin (Vector) in 2 M potassium acetate. Cerebrospinal fluid was drained via a cisternal puncture to reduce brain pulsation and the animal was suspended by a clamp applied to the tail. After placement of a microelectrode in the cortex above the hippocampus for recording, the exposed surface of the brain was covered with soft paraffin wax. The microelectrode was advanced slowly into the hippocampus to impale CA1 neurons. After impalement, the neurons with a stable membrane potential of ⫺60 mV or greater were selected for further study. Data were digitized and stored on a Macintosh computer using the data acquisition program Axodata (Axon Instruments). The Student’s t-test or ANOVA was used for statistical analysis (Staview, Abacus Concepts). After each successful recording, neurobiotin was
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Fig. 2. Spontaneous activity of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. Before ischemia, the CA1 neuron exhibited active spontaneous membrane potential fluctuation. The action potential in this trace is truncated. After ischemia, spontaneous firing of all neurons disappeared. The spontaneous synaptic activity in L-PSP neurons decreased significantly by 3 h and ceased by 41 h following reperfusion. However, the non-L-PSP neurons still maintained spontaneous synaptic activity at this time. No spontaneous synaptic activity was observed in S-EPSP neurons. The scales apply to all recordings.
iontophoresed into the cell by passing depolarizing current pulses (2 Hz, 300 ms, 0.5–1 nA) for 10 min. At the end of the experiment, the animal was deeply anesthetized and perfused transcardially with 0.01 M phosphate-buffered saline followed by 4% paraformaldehyde. The brain was removed and stored in fixative overnight. Coronal sections were cut at 50 mm thickness using a Vibratome and incubated in 0.1% horseradish peroxidase-conjugated avidin-D (Vector) in 0.01 M potassium phosphate-buffered saline (pH 7.4) with 0.5% Triton X-100 overnight at room temperature. After detection of peroxidase activity with 3,3 0 -diaminobenzidine, sections were examined in potassium phosphatebuffered saline. Sections containing labeled neurons were mounted on gelatin-coated slides and counterstained with Cresyl Violet for light microscopy.
RESULTS
Experiments were performed on control rats (n 10) and rats subjected to forebrain ischemia with ischemic depolarization of 13.8 ^ 0.9 min (n 37). This degree and duration of ischemia consistently produced selective cell death in the CA1 region of rat hippocampus using the fourvessel occlusion method under halothane anesthesia. 21 The arterial gas tension (pco2, po2, pH, glucose and hemoglobin) and general physiological condition (arterial blood pressure, rectal and brain temperature) of the animals before and after ischemia did not differ and were maintained within
normal ranges throughout the experiments. A total of 128 neurons was analysed in the present study, of which 110 were successfully stained and identified as CA1 pyramidal cells. The 18 unidentified cells, considered as CA1 pyramidal neurons based on stereotaxic parameters and electrophysiological characteristics, were also included in the present study. The control data presented in this report were derived from 22 neurons recorded before ischemia. Intracellular recording of CA1 neurons was very difficult two days after ischemia, especially in the medial portion of the CA1 zone. Among 10 animals in which recordings were attempted during 37–48 h after reperfusion, five showed unequivocal cell degeneration in the medial protion of the CA1 zone with hematoxylin–eosin staining. However, no gross signs of degeneration, such as swelling, shrinkage of cell body or fragmentation of dendrites, were observed in postischemic neurons having undergone intracellular recording and labeled with neurobiotin. In a previous report, 7 we identified three distinct populations of CA1 pyramidal neurons after reperfusion based on difference in synaptic response, and named these late depolarizing postsynaptic potential (L-PSP), non-L-PSP and small excitatory postsynaptic potential (S-EPSP) neurons (Fig. 1). In order to study the temporal changes of membrane properties following reperfusion, the
⫺73 ⫺73 ⫺70 ⫺70 ⫺69
^ 6.28 (22) ^ 4.75 (11) ^ 5.41 (22) ^ 6.21 (18) ^ 4.82 (12)
RMP (mV) 77 81 86 79 79
^ 7.52 (22) ^ 12.09 (10) ^ 10.47** (20) ^ 9.70 (13) ^ 7.73 (9)
Spike height (mV) 0.82 ^ 0.20 (22) 0.96 ^ 0.24 (10) 0.85 ^ 0.15 (20) 1.07 ^ 0.51* (13) 0.74 ^ 0.16 (9)
Spike width (ms) 25.63 ^ 5.51 (15) 22.04 ^ 9.25 (7) 22.02 ^ 6.22 (16) 14.80 ^ 8.84** (9) 16.68 ^ 7.20** (9)
Rin (MV) 12.44 ^ 2.85 (15) 9.87 ^ 2.97 (7) 12.53 ^ 4.99 (16) 6.98 ^ 3.63** (9) 7.94 ^ 2.52** (9)
Time constant (ms)
0.53 ^ 0.58 (16) 0.00 ^ 0.00** (11) 0.02 ^ 0.11** (22) 0.00 ^ 0.01** (18) 0.00 ^ 0.00** (12)
Spont. firing (Hz)
77 ^ 7.52 (22) 77 ^ 10.14 (11) 74 ^ 8.34 (3) 84 ^ 7.00 (7) 78 ^ 11.87 (6)
⫺73 ^ 6.28 (22) ⫺69 ^ 7.06 (13) ⫺67 ^ 3.17 (4) ⫺69 ^ 4.12 (7) ⫺69 ^ 2.38 (7)
Before ⬃ 5 h after ⬃ 19 h after ⬃ 31 h after ⬃ 42 h after
0.82 ^ 0.20 (22) 0.84 ^ 0.11 (11) 0.76 ^ 0.16 (3) 0.89 ^ 0.12 (7) 0.69 ^ 0.09 (6)
Spike width (ms) 25.63 ^ 5.51 (15) 22.94 ^ 7.68 (12) 24.23 ^ 5.93 (3) 28.68 ^ 3.45 (5) 20.25 ^ 3.90 (4)
Rin (MV)
12.44 ^ 2.88 (15) 14.25 ^ 6.03 (12) 13.50 ^ 3.64 (3) 10.96 ^ 2.04 (5) 7.98 ^ 0.53 (4)
Time constant (ms)
0.53 ^ 0.58 (16) 0.05 ^ 0.14** (13) 0.03 ^ 0.07** (4) 0.03 ^ 0.07** (7) 0.00 ^ 0.03** (7)
Spont. firing (Hz)
77 ^ 7.52 (22) 78 ^ 12.11 (5) 77 ^ 16.26 (3)
⫺73 ^ 6.28 (22) ⫺71 ^ 7.21 (7) ⫺69 ^ 5.32 (4)
Before ⬃ 31 h after ⬃ 42 h after
0.82 ^ 0.20 (22) 0.85 ^ 0.20 (5) 0.75 ^ 0.21 (3)
Spike width (ms)
25.63 ^ 5.51 (15) 15.73 ^ 11.34* (4) 12.50 ^ 10.54* (3)
Rin (MV)
12.44 ^ 2.88 (15) 7.35 ^ 3.49** (4) 6.57 ^ 6.03** (3)
Time constant (ms)
0.53 ^ 0.58 (16) 0.00 ^ 0.00** (7) 0.01 ^ 0.03** (4)
Spont. firing (Hz)
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. *P ⬍ 0.05, **P ⬍ 0.01, ANOVA.
Spike height (mV)
RMP (mV)
Reperfusion
Table 3. Membrane properties of small excitatory postsynaptic potential neurons at different periods after ischemia
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. **P ⬍ 0.01, ANOVA.
Spike height (mV)
RMP (mV)
Reperfusion
Table 2. Membrane properties of non-late depolarizing postsynaptic potential neurons at different periods after ischemia
Values are means ^ S.D., with number of neurons in parentheses. Spike height is measured from the resting membrane potential. Spike width is measured at half of the peak amplitude of the action potential. Input resistance (Rin) is derived from the linear portion of the current–voltage curve (0 to ⫺ 0.5 nA). Time constant is derived from transients of hyperpolarizing pulse ( ⫺ 0.3 nA, 200 ms). RMP, resting membrane potential. *P ⬍ 0.05, **P ⬍ 0.01, ANOVA.
Before ⬃5 h after ⬃19 h after ⬃31 h after ⬃42 h after
Reperfusion
Table 1. Electrophysiological properties of late depolarizing postsynaptic potential neurons at different periods after reperfusion
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Fig. 3. Current–voltage (I–V) relationship of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. Each trace is the average of four recordings. Top panels are membrane potential deflections caused by the current pulses. Bottom panels are intracellularly applied current pulses. (A) I–V relationship before ischemia. A marked anomalous rectification was observed in this neuron. (B–D) I–V relationship of representative L-PSP, non-L-PSP and S-EPSP neurons, respectively. Scales in D apply to A–C. The action potentials in these figures are truncated. (E–G) Plots of I–V curve from control and three groups of neurons, showing the input resistance changes at different intervals after reperfusion. Beginning on the second day following reperfusion, the slope of the I–V curve of L-PSP neurons (E) and S-EPSP neurons (G) was persistently smaller than that of controls, indicating the reduction in input resistance. At the same time, the inward rectification with depolarizing currents changed to an outward rectification in these two groups of neurons and this outward rectification increased progressively after reperfusion. The I–V curves of non-L-PSP neurons at different intervals after reperfusion were about the same as before ischemia (F).
neurons within these three groups were pooled according to the reperfusion time at which they were recorded: 0–12 h (median: 5 h); 12–24 h (median: 19 h); 24– 36 h (median: 31 h); 36–48 h (median: 42 h). Spontaneous activity after reperfusion Before ischemia, the spontaneous firing rate of CA1 pyramidal neurons was 0.53 ^ 0.58 spikes/s (n 16). Spontaneous neuronal firing ceased shortly after ischemia in all three groups of neurons and remained markedly suppressed up to 48 h following reperfusion (Tables 1–3). Neuronal hyperactivity as
indicated by increased spontaneous firing rate was never observed in the present study. Spontaneous synaptic activity as indicated by small PSPs superimposed on baseline membrane potentials was very active in CA1 neurons before ischemia. Spontaneous synaptic activity in L-PSP and non-L-PSP neurons decreased significantly 3 h after reperfusion and was completely abolished 36–48 h after reperfusion in L-PSP neurons (Fig. 2). Spontaneous synaptic activity in non-L-PSP neurons, however, remained at low levels 36–48 h after reperfusion. No spontaneous synaptic activities were observed in S-EPSP neurons.
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passive membrane conductance, the active membrane conductance of CA1 neurons also changed remarkably after ischemia. As shown in Fig. 3, CA1 neurons before ischemia exhibited an obvious inward rectification when membrane potential was maintained at depolarizing or hyperpolarizing extremes by current injection. The latter results are similar to previous observations. 11,20 After ischemia, the inward rectification with depolarizing currents changed to an outward rectification in both L-PSP and S-EPSP neurons, and this outward rectification increased progressively following reperfusion (Fig. 3E, G). No such change was observed in non-L-PSP neurons (Fig. 3F). The alteration of inward rectification with hyperpolarizing currents in all three groups of neurons was not as great as that in the depolarizing extreme (Fig. 3E–G). The time constant of CA1 neurons after reperfusion showed similar changes as those of input resistance. The time constant of L-PSP and S-EPSP neurons decreased to approximately 50% of preischemic values 31–42 h after reperfusion (P ⬍ 0.01, ANOVA; Tables 1 and 3). In non-L-PSP neurons, the time constant remained normal until after ⬃31 h and then declined at 42 h following reperfusion (Table 2). Excitability, repetitive firing and fast afterhyperpolarization after reperfusion Fig. 4. Plots showing the changes in spike threshold (A) and rheobase (B) of CA1 pyramidal neurons at different intervals after reperfusion. The value of each plot is the mean, with the number of neurons in parentheses. The spike threshold and rheobase of L-PSP and S-EPSP neurons increased gradually following reperfusion, indicating a progressive decrease in neuronal excitability. However, a transient increase in spike threshold and rheobase was observed in non-L-PSP neurons at the early reperfusion period.
Input resistance and time constant after reperfusion To compare the current–voltage relationship of CA1 neurons before and after ischemia, constantcurrent pulses (200 ms, ⫺1.0 to ⫹0.5 nA) were passed; the voltage values were measured from the averages of four recordings at the steady state of the transients (average between 160 and 180 ms from the beginning of the pulse). The membrane input resistance of CA1 neurons was 25.63 ^ 5.51 MV (n 15) in control animals. The input resistance of L-PSP neurons remained unchanged during the first 24 h after reperfusion, but decreased significantly to 14.80 and 16.68 MV at 31 and 42 h after reperfusion, respectively (P ⬍ 0.01, ANOVA; Table 1). The input resistance of non-L-PSP neurons, in contrast, displayed no significant changes up to 48 h following reperfusion (Table 2). The input resistance of S-EPSP neurons was decreased significantly, to 15.73 and 12.50 MV at 31 and 42 h after reperfusion, respectively (P ⬍ 0.05, ANOVA; Table 3). In addition to the dramatic alterations in
The changes in neuronal excitability were evaluated by comparing the spike threshold and rheobase of CA1 neurons before and after ischemia. The spike threshold was measured at the beginning of the upstroke of the first action potential and the rheobase was determined as the minimal intensity of depolarizing current pulse to trigger an action potential. As shown in Fig. 4, the spike threshold and rheobase of L-PSP and S-EPSP neurons increased progressively after reperfusion, while only a transient increase in spike threshold (P ⬍ 0.05, ANOVA) and rheobase (P ⬍ 0.01, ANOVA) was detected in non-L-PSP neurons at 5 h after reperfusion. Repetitive firing was induced by application of depolarizing current pulses (400 ms, 0.1–1.6 nA). In control neurons, there was a strong spike frequency adaptation of spike trains during application of constant-current pulses, as indicated by the differences in spike frequency between the first interspike interval (ISI) and the second ISI (Fig. 5). The repetitive firing of L-PSP neurons was dramatically depressed after ischemic insult. The spike frequency declined progressively at all current intensities, with the lowest firing rate at 42 h after reperfusion (Fig. 5D). In contrast, the spike frequency of non-L-PSP neurons remained about the same as that of controls, except the frequency of the second ISI increased at stronger depolarizing current pulses following reperfusion (Fig. 5E). In CA1 pyramidal neurons, each action potential is followed by a fast afterhyperpolarization (fAHP),
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Fig. 5. Repetitive firing of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. (A–C) Spike trains were evoked by 0.6-nA depolarizing current pulses. Examples of repetitive firing of a control neuron (A), an L-PSP neuron 34 h after reperfusion (B) and a non-L-PSP neuron 32 h after reperfusion (C). Scales in B apply to A and C. (D, E) Plots showing the changes in the relationship between depolarizing current and spike frequency of the first ISI (upper panel) and second ISI (lower panel) of L-PSP and non-L-PSP neurons at different intervals after reperfusion. Each point is the mean value. The spike frequency increased linearly with the increase of current intensities. In comparison with control values, the spike frequency of both the first and second ISIs decreased progressively in L-PSP neurons following reperfusion, indicating a gradual suppression of repetitive firing. However, in non-L-PSP neurons, the spike frequency of the first ISI remained unchanged after reperfusion, although there was an increase in the spike frequency of the second ISI at high current intensities.
which is mediated by calcium-activated potassium current. 15,23 In the present study, the fAHP was induced by the injection of a depolarizing current pulse that generates action potentials. The amplitude of the fAHP was measured as deviation from the beginning of the upstroke of an action potential within 5 ms after the peak of a single spike. The amplitude of the fAHP increased significantly, from around 1 mV in control neurons to approximately 5 mV in L-PSP neurons (P ⬍ 0.01, ANOVA). The amplitude of the fAHP in L-PSP neurons at different intervals following reperfusion was slightly different, with the highest value at ⬃ 5 h and the lowest value at ⬃ 31 h after reperfusion (Fig. 6E). Although the amplitude of the fAHP in non-L-PSP neurons also increased after reperfusion and showed a similar pattern to that of L-PSP neurons, the amplitude of the fAHP in non-L-PSP neurons was approximately 2 mV smaller than that of L-PSP neurons at each reperfusion interval (Fig. 6E). The amplitude of the fAHP in S-EPSP neurons was the same as that in L-PSP neurons. DISCUSSION
The present study demonstrates that the postischemic membrane properties differ among the three groups of CA1 neurons distinguished by their
postischemic synaptic transmission properties (i.e. L-PSP, non-L-PSP and S-EPSP neurons). 7 The differences in synaptic transmission and membrane properties of these postischemic neurons suggest that they may have different histopathological outcomes. Alteration of input resistance following reperfusion In agreement with previous studies, 13 we observed that the membrane input resistance of CA1 neurons within 24 h reperfusion remained similar to control neurons. However, after 24 h reperfusion, the input resistance declined significantly in LPSP and S-EPSP neurons, but did not change in nonL-PSP neurons. The decrease of input resistance in L-PSP and S-EPSP neurons 24–48 h after reperfusion differs from the results of previous studies. It has been reported that no difference was found in the input resistance of CA1 neurons between control hippocampal slices and slices prepared 24 or 48 h after 20-min ischemia. 10,13 It is difficult to reconcile that membrane input resistance of CA1 neurons 24– 48 h after reperfusion remains normal, while morphological signs of cell degeneration were already apparent. 10 The reduced input resistance observed in L-PSP and S-EPSP neurons in the present study may be due to the loss of functional membrane integrity and disruption of ion conductance
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associated with pathological changes after ischemia. The unchanged input resistance of non-L-PSP neurons, however, suggests that these neurons may survive the ischemic insult or undergo degenerating pathways different from those of L-PSP and S-EPSP neurons. Depression of neuronal excitability following reperfusion
Fig. 6. Fast afterhyperpolarization (fAHP) of CA1 pyramidal neurons before ischemia and at different intervals after reperfusion. fAHPs were induced by action potentials evoked by a depolarizing current pulse. (A–D) The amplitude of the fAHP increased in all three groups of postischemic neurons. Scales in D apply to A–C. The action potentials in these figures are truncated. (E) Plots showing the changes in amplitude of the fAHP in CA1 neurons after reperfusion. Each point is the mean value. In L-PSP and S-EPSP neurons, the amplitude of the fAHP was persistently larger than that of control neurons (P ⬍ 0.01, ANOVA) during the whole course of reperfusion. Although the non-L-PSP neurons showed similar changes to those of LPSP neurons, the amplitude of the fAHP in these neurons was obviously lower than those in L-PSP and S-EPSP neurons at each corresponding reperfusion time.
The gradual increase in the spike threshold in LPSP and S-EPSP neurons indicates that neuronal excitability in these cells is progressively depressed following reperfusion. In contrast, only a transient increase in spike threshold was observed in non-LPSP neurons. Using the gerbil brain slice preparation at different time-points after ischemia, Urban et al. 25 reported that the excitability of CA1 neurons was depressed in a similar time-dependent fashion. The depression of neuronal excitability may be responsible for the decrease in spontaneous firing rate of CA1 neurons following ischemia, as observed in the present study and in previous reports. 2,6,12,27 Increased spike threshold after reperfusion accounts for many postischemic changes observed in the present study. For example, the increased rheobase after reperfusion is closely related to the increase in spike threshold and it may also contribute to the depression of repetitive firing after reperfusion. Increased spike threshold after reperfusion has also been observed in spiny neurons in the neostriatum. 26 The mechanism underlying increased spike threshold after reperfusion in neostriatal neurons may reflect dopamine effects on D1 dopamine receptors. 26 It has been shown that decreased excitability of CA1 neurons during anoxia is due to the increase in potassium conductance. 5,9,16 The mechanisms causing the increase of spike threshold in CA1 neurons after reperfusion are not clear at this point. Two different types of changes in neuronal excitability were observed in CA1 neurons after reperfusion: a progressive increase in spike threshold was detected in L-PSP and S-EPSP neurons, while a transient increase in spike threshold was observed in non-L-PSP neurons and in CA1 neurons following 5-min ischemia. 27 Since ischemia of 5 min duration will not cause neuronal degeneration in the rat hippocampus, the transient increase in spike threshold is probably a compensatory response to the ischemic stress rather than an event associated with the process of cell death. The progressive increase in spike threshold in L-PSP and S-EPSP neurons, however, may be a sign of membrane degradation following ischemia. The progressive decrease in input resistance in these neurons also supports this notion. CONCLUSIONS
The differences in membrane properties between L-PSP and non-L-PSP neurons following ischemia
Membrane properties of CA1 neurons after ischemia
strongly indicate that these two groups of neurons behave differently to severe ischemia. L-PSP neurons show an enhanced synaptic transmission and a progressive decrease in excitability following reperfusion, while non-L-PSP neurons show a depressed synaptic transmission and transient decrease in excitability following ischemia. During the first 12 h of reperfusion, the synaptic transmission is enhanced in L-PSP neurons and depressed in non-L-PSP neurons. 7 However, the reduced excitability of both groups of neurons with an unchanged input resistance suggests that the postischemic changes in synaptic transmission in CA1 neurons are not due to the alterations in their membrane properties. The late but progressive decrease in
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input resistance in L-PSP and S-EPSP neurons supports the notion that these neurons are the degenerating neurons following severe ischemic insult. In contrast, the transient decrease in excitability and unchanged input resistance following reperfusion in non-L-PSP neurons suggest that they may have different postischemic outcome to that of L-PSP and S-EPSP neurons.
Acknowledgements—This research was supported by grants AHA 94008130, NIH NS33101 to Z.C.X. and SemmesMurphey Chair of Excellence to W.A.P. T.M.G. is the recipient of postdoctoral fellowships from the Neuroscience Center of Excellence in U.T. Memphis and grants NNSF 39570263 and NSFGP 950537 of P. R. China.
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