Prolonged enhancement and depression of synaptic transmission in CA1 pyramidal neurons induced by transient forebrain ischemia in vivo

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PII:

Neuroscience Vol. 87, No. 2, pp. 371–383, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(98)00150-X

PROLONGED ENHANCEMENT AND DEPRESSION OF SYNAPTIC TRANSMISSION IN CA1 PYRAMIDAL NEURONS INDUCED BY TRANSIENT FOREBRAIN ISCHEMIA IN VIVO T.-M. GAO,*† W. A. PULSINELLI* and Z. C. XU*‡§ *Department of Neurology, University of Tennessee at Memphis, Memphis, TN 38163, U.S.A. †Department of Physiology, The First Military Medical University, Guangzhou, People’s Republic of China Abstract––Evoked postsynaptic potentials of CA1 pyramidal neurons in rat hippocampus were studied during 48 h after severe ischemic insult using in vivo intracellular recording and staining techniques. Postischemic CA1 neurons displayed one of three distinct response patterns following contralateral commissural stimulation. At early recirculation times (0–12 h) approximately 50% of neurons exhibited, in addition to the initial excitatory postsynaptic potential, a late depolarizing postsynaptic potential lasting for more than 100 ms. Application of dizocilpine maleate reduced the amplitude of late depolarizing postsynaptic potential by 60%. Other CA1 neurons recorded in this interval failed to develop late depolarizing postsynaptic potentials but showed a modest blunting of initial excitatory postsynaptic potentials (non-late depolarizing postsynaptic potential neuron). The proportion of recorded neurons with late depolarizing postsynaptic potential characteristics increased to more than 70% during 13–24 h after reperfusion. Beyond 24 h reperfusion, 20% of CA1 neurons exhibited very small excitatory postsynaptic potentials even with maximal stimulus intensity. The slope of the initial excitatory postsynaptic potentials in late depolarizing postsynaptic potential neurons increased to 150% of control values up to 12 h after reperfusion indicating a prolonged enhancement of synaptic transmission. In contrast, the slope of the initial excitatory postsynaptic potentials in non-late depolarizing postsynaptic potential neurons decreased to less than 50% of preischemic values up to 24 h after reperfusion indicating a prolonged depression of synaptic transmission. More late depolarizing postsynaptic potential neurons were located in the medial portion of CA1 zone where neurons are more vulnerable to ischemia whereas more non-late depolarizing postsynaptic potential neurons were located in the lateral portion of CA1 zone where neurons are more resistant to ischemia. The result from the present study suggests that late depolarizing postsynaptic potential and small excitatory postsynaptic potential neurons may be irreversibly injured while non-late depolarizing postsynaptic potential neurons may be those that survive the ischemic insult. Alterations of synaptic transmission may be associated with the pathogenesis of postischemic neuronal injury.  1998 IBRO. Published by Elsevier Science Ltd. Key words: ischemia, hippocampus, long-term potentiation, long-term depression, excitotoxicity, in vivo intracellular recording.

CA1 pyramidal neurons in hippocampus are highly vulnerable to cerebral ischemia. Brief periods (min) of severe ischemia cause neuronal degeneration in the CA1 region of hippocampus but such injury only ‡To whom correspondence should be addressed. §Present address: Department of Anatomy, Indiana University Medical Center, Indianapolis, IN 46202, U.S.A. Abbreviations: AMPA, á-amino-3-hydroxy-5-methyl-4isoxazolepropionate; CaBP, calbindin-D28k; CC, contralateral commissural pathway; DAB, 3 ,3 diaminobenzidine; EPI, EPSP paired-pulse facilitation index; EPSP, excitatory postsynaptic potential; fAHP, fast afterhyperpolarization; IPSP, inhibitory postsynaptic potential; KPBS, potassium phosphate-buffered saline; L-PSP, late depolarizing postsynaptic potential; LTD, long-term depression; LTP, long-term potentiation; MK801, dizocilpine maleate; NBQX, 3-dihydroxy-6-nitro-7sulfamoyl-benzo[f]quinoxaline; NMDA, N-methyl-aspartate; PBS, phosphate-buffered saline; PPF, pairedpulse facilitation.

becomes histologically evident three to seven days after recirculation.41,60,61 Excessive accumulation of extracellular glutamate and Ca2+ influx during ischemia have been postulated to cause neuronal hyperactivity and trigger neuronal injury.12,64 However, the potentially toxic levels of extracellular glutamate and Ca2+ influx are short-lived, returning to normal within 30 min after cerebral reperfusion.6,52 This brief exposure to excessive glutamate and Ca2+ is difficult to reconcile with the onset of CA1 neuronal death that manifests itself days later. Moreover, in vivo electrophysiological studies of the postischemic hippocampus provide conflicting results, with some data showing an increase10,72 and other data a decrease9,24,37 of the CA1 neuronal firing rate in the early hours after transient ischemia. Results from hippocampal slice preparations subjected to various hypoxic/ischemic paradigms provide similar conflicting results on synaptic transmission in

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postischemic CA1 neurons. Some studies indicated that the synaptic transmission was unchanged38 or suppressed4,23,30 following hypoxia/ischemia. Other studies, in contrast, reported an enhancement of synaptic transmission after hypoxia/ ischemia.16,35,42,75,76 The data from in vitro studies are further compromised by the uncertainty of whether the slice preparation accurately reflects the in vivo pathophysiology of delayed ischemic brain injury. Delayed ischemic injury to hippocampal neurons in the animal model used in the present study can be attenuated by á-amino-3-hydroxy-5-methyl-4isoxazolepropionate (AMPA) receptor blockers.7,68 This antagonist has protective effects on CA1 neurons even when administrated hours after brief cerebral ischemia.7,56 While prolonged reduction of body temperature may contribute to such protective effects,57 these results suggest that such intervention may be protective even hours to days after the resolution of the ischemia-induced rise in extracellular glutamate levels. A recent in vivo study from this laboratory has demonstrated a late depolarizing postsynaptic potential (L-PSP) induced from CA1 pyramidal neurons after transient ischemia indicating an enhancement of synaptic transmission following ischemia.25 To further clarify the possibility of latedeveloping alterations of synaptic transmission following ischemia, we present results from in vivo intracellular recordings in postischemic CA1 hippocampal neurons. Our objective was to document in vivo electrophysiological changes in synaptic efficacy in neurons destined to die from a predictably lethal ischemic insult and to determine when in the temporal profile of cell death such changes occurred. Preliminary results from these studies were presented in abstract form.26 EXPERIMENTAL PROCEDURES

Transient forebrain ischemia NIH guidelines for the care and use of laboratory animals (NIH Publications No. 80–23) were strictly followed. Male adult Wistar rats (250–350 g, Sasco) were subjected to transient forebrain ischemia using the method of four-vessel occlusion59 with some modifications. Food but not water was withheld overnight to provide uniform blood glucose levels. The animals were anesthetized with 1–2% halothane mixed with 33% O2 and 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 of the animal. A caudal artery was cannulated for arterial blood pressure monitoring and sampling for blood gas measurements. A femoral vein was cannulated in a group of animals for administration of dizocilpine maleate (MK-801; 2.0 mg/kg; Research Biochemicals International). The animals were then placed in a stereotaxic frame and the core body temperature was maintained at 37C 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 tempera-

ture was maintained at 37C 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 The extracellular d.c. potential in CA1 region of hippocampus was monitored during ischemia. Recording microelectrodes were pulled from glass capillaries with filaments (A-M system) to a tip resistance of 5 MÙ 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 hippocampus (2.5 mm below brain surface). Following baseline recordings, forebrain ischemia was initiated by tightening the occluding device. Cerebral blood flow was typically reduced to less than 10% of the control levels based on laser Doppler recordings from cortex. Ischemic depolarization, as indicated by d.c. potential shifts from 0 mV to
20 mV, occurred approximately 2.5 min after occlusion. Carotid arteries were released at about 12 min after the onset of ischemic depolarization, after which blood flow resumed immediately and the d.c. potential returned to 0 mV in approximately 2 min. The duration of ischemic depolarization was measured from the onset of negative deflection of d.c. potential to the point at which the d.c. potential completely returned to pre-ischemic levels after recirculation. Only animals with ischemic depolarization of 14 min were used for electrophysiological experiments. For intracellular recordings later than 12 h after reperfusion, the animals were returned to their cages 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 Intracellular recording and staining were performed immediately after ischemia and up to 48 h after reperfusion. Preparation for recording was as previously described.79,81 Briefly, under 1–2% halothane anesthesia, the skull was opened to expose the recording site and for placement of stimulus electrodes. A pair of bipolar stimulating electrodes were placed into the contralateral commissural pathway (AP 3.7–4.7 mm, ML 1.5 mm, DV 3.5 mm) and were used to deliver constant current pulses of 0.1 ms duration at various intensities. Recording electrodes were pulled from glass capillaries with filaments to a tip resistance of 50– 80 MÙ 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 (AP 3.0–6.5 mm, ML 1.0–4.0 mm), the exposed surface of the brain was covered with soft paraffin wax. After impalement, neurons with stable membrane potentials of
60 mV or greater were selected for further study. Data were digitized and stored with a Macintosh computer using the data acquisition program Axodata (Axon Instruments). Student t-test or ANOVA (Fisher’s PLSD) were used for most of the statistical analysis in the present study except ÷2-test was used for analysing cell localization (StatView, Abacus Concepts). After each successful recording neurobiotin was iontophoresed into the cell by passing a depolarizing current pulse (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 (PBS) followed by 4% paraformaldehyde. The brain was removed and stored in fixative overnight. Coronal sections were cut at 50 µm thickness using a Vibratome, and incubated in 0.1% horseradish peroxidase-conjugated avidin-D (Vector) in 0.01 M potassium phosphate-buffered saline (KPBS, pH 7.4) with 0.5% Triton X-100 overnight at room temperature.

Synaptic transmission in CA1 neurons after ischemia After detection of peroxidase activity with 3 ,3 diaminobenzidine (DAB), sections were examined in KPBS. Sections containing labeled neurons were mounted on gelatin-coated slides and counterstained with Cresyl Violet for light microscopy. RESULTS

Experiments were performed on 47 rats, of which 10 served as control animals while 37 were subjected to forebrain ischemia that resulted in ischemic depolarization of 13.80.9 min (MeanS.D.). Ischemia of this degree and duration consistently produced selective cell death in the CA1 region of rat hippocampus using the four-vessel occlusion method under halothane anesthesia.62 The arterial gas tension (PCO2, PO2), pH, glucose, Hgb, arterial pressure and the rectal/brain temperatures of these animals were maintained within normal ranges throughout the experiments. A total of 163 neurons were analysed, with 137 stained successfully and identified as CA1 pyramidal cells. No gross evidence of degeneration such as shrinkage, swollen somata or dendritic fragmentation were observed in these neurons. The 26 unidentified neurons were considered as CA1 pyramidal neurons based on stereotaxic parameters and neurophysiological characteristics and were included in the study. Intracellular recording of CA1 neurons became extremely difficult two days after reperfusion, 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 portion of CA1 zone with Hematoxylin and Eosin staining. We therefore focused on the changes of synaptic transmission of CA1 neurons during the first 48 h following reperfusion. Electrophysiological responses in postischemic CA1 neurons In control animals, stimulation of the contralateral commissural pathway (CC) elicited excitatory postsynaptic potentials (EPSPs) from CA1 pyramidal neurons followed by inhibitory postsynaptic potentials (IPSPs) (Fig. 1A). The initial EPSPs triggered action potentials when stimulus intensities were strong enough. The same stimuli elicited three different responses from CA1 pyramidal neurons after ischemia. Approximately 60% of the neurons examined (83/140) demonstrated a late depolarizing postsynaptic potential (L-PSP) in addition to the initial EPSP (Fig. 1B). The L-PSPs were elicited as early as 4 h and as late as 48 h after reperfusion. More than 30% of the neurons (41/140) failed to develop L-PSPs upon CC stimulation and exhibited moderately attenuated synaptic responses that were otherwise comparable to those in control animals (Fig. 1C). A third, smaller group of neurons (16/140) also failed to develop L-PSPs but the synaptic responses of these neurons were more severely

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attenuated (Fig. 1D). These three groups were designated as L-PSP, Non L-PSP and S-EPSP neurons, respectively. The relationship between stimulus intensity and the amplitude of initial EPSP differed among these groups, with L-PSP neurons showing increased amplitude while Non L-PSP and S-EPSP neurons showing reduced amplitude across the range of stimulus intensities tested (Fig. 1E). To study the dynamics of electrophysiological responses after reperfusion, the neurons were pooled according to the reperfusion time during which they were recorded: 0–12 h (median: 5 h); 13–24 h (median 19 h); 25–36 h (median: 31 h); 37–48 h (median: 42 h). The number of L-PSP and Non L-PSP neurons was approximately equal through 12 h after reperfusion (Fig. 1F). The proportion of L-PSP neurons increased to more than 70% during 13–24 h and declined slightly thereafter, while S-EPSP neurons composed 20% of the population after 24 h. In contrast, the percentage of Non L-PSP neurons progressively decreased through 36 h. It should be noted that during 37–48 h after reperfusion it was difficult to record from the medial portion of CA1 zone because most neurons in this region had degenerated. The properties of late depolarizing postsynaptic potentials Paired-pulse stimulation with an interstimulus interval of 50 ms remarkably potentiated the L-PSP, and action potentials were triggered from the second L-PSP (Fig. 2A, B). Occasionally, a single stimulus could also evoke action potentials from the L-PSP (Fig. 2C). No significant changes in the amplitude of L-PSPs were found through 48 h reperfusion while the duration of L-PSPs in 19 h group was significantly longer than those observed at later intervals (Table 1). The late-developing and long-lasting nature of L-PSPs suggested the involvement of N-methyl-aspartate (NMDA) receptor-mediated events.13,32 In five animals (one cell per animal) subjected to ischemia, intravenous administration of the NMDA receptor antagonist MK-801 (2.0 mg/kg) remarkably attenuated the amplitude and duration of L-PSPs (Fig. 2D). The amplitude of L-PSPs was reduced to 40% of control values at 15–20 min (P<0.01, t-test), then returned to the control levels at 30–50 min after application of MK-801. MK-801 also suppressed the amplitude of the initial EPSPs but to a lesser degree than that of L-PSPs. The effect of MK-801 on the amplitude and duration of L-PSPs and initial EPSPs is shown in Fig. 2E. Prolonged synaptic enhancement and depression after ischemia The characteristics of PSPs evoked by CC stimulation at 2.5 times the threshold stimulus intensity were compared before ischemia and at intervals

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Fig. 1. Evoked synaptic responses from CA1 neurons before and after transient forebrain ischemia. (A) Recording from a control rat in which stimulation of contralateral commissural pathway (CC) elicited an EPSP followed by an IPSP. An action potential was triggered from the EPSP. (B) After ischemia, the same stimulus evoked an additional late depolarizing postsynaptic potential (L-PSP) from 60% of the CA1 neurons. Action potentials were occasionally triggered from L-PSPs. (C) In 30% of the neurons, CC stimuli elicited a EPSP/IPSP sequence with smaller amplitude in comparison with the control neurons. (D) Approximately 31 h after reperfusion, a small EPSP was evoked from 10% of the neurons even with maximal stimulus intensity. Traces in B, C and D were recorded 27 h, 35 h and 28 h after reperfusion, respectively. Each trace is the average of four recordings. The action potentials in these figures are truncated. The scales in A apply to B–D. (E) The relationship between stimulus intensity and the amplitude of initial EPSPs in CA1 neurons before and after ischemia. In comparison with neurons before ischemia, the amplitude of EPSPs in L-PSP neurons was larger and that in Non L-PSP neurons was smaller. In S-EPSP neurons, the amplitude of EPSPs was about the same with increasing stimulus intensities. The values in the plotting are means. The data for L-PSP and Non L-PSP neurons was collected at 5 h after reperfusion and for S-EPSP neurons was collected at 31 h after reperfusion. The stimulus intensities are the magnitudes of the threshold (T) for inducing the initial EPSP. (F) The proportion of three distinct responses in CA1 neurons at different periods following reperfusion. The percentage of L-PSP neurons gradually increased and peaked at 19 h, while the percentage of Non L-PSP neurons progressively decreased following reperfusion. The number of recorded neurons is in parenthesis.

after reperfusion. The initial EPSPs were characterized by their amplitude and slope (measured from 10% to 90% of the maximal amplitude), duration (measured at 50% of maximal amplitude), threshold and latency. The slope of initial EPSPs in L-PSP neurons increased to 155% that of control neurons (P<0.05, ANOVA) around 5 h after reperfusion (Fig. 3A), suggesting that a prolonged synaptic facilitation was induced in some CA1 neurons by transient ischemia. The slope of initial EPSPs returned to the preischemic levels 19 h after reperfusion. In contrast, the slope of initial EPSPs in Non L-PSP neurons progressively decreased to as low as 40% of control levels around 19 h after

reperfusion (P<0.01, Fig. 3A) suggesting that ischemia induced a prolonged synaptic depression in this subgroup of CA1 neurons. The slope of initial EPSPs in S-EPSP neurons was dramatically depressed at all times (P<0.01, Fig. 3A). These changes in slope were consistent with the effects on the amplitude of initial EPSPs of all three types of neurons (data not shown). The duration of initial EPSPs was progressively longer in L-PSP neurons and shorter in Non L-PSP neurons than observed in control neurons (Fig. 3B). The stimulus threshold for inducing initial EPSPs was significantly reduced in both L-PSP and Non L-PSP neurons (Fig. 3C), but the latency of EPSPs in these neurons was about the

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Fig. 2. The properties of L-PSPs. (A) In most cases, the L-PSP induced by single stimulus could not trigger an action potential. (B) Paired-pulse stimulation with interstimulus interval of 50 ms remarkably enhanced the amplitude and duration of L-PSP and produced a burst of action potentials. A and B are recordings from the same neuron. (C) A single stimulus occasionally triggered a burst of firing from L-PSPs. (D) Recordings before and after application of MK-801. The amplitude and the duration of L-PSP and the initial EPSP were reduced 15 min after injection of MK-801. The effect of MK-801 lasted less than 30 min. C and D are the average of four recordings. The action potentials in these figures are truncated. The scales in C apply to A and B. (E) Histograms showing the effect of MK-801. The amplitude and duration of L-PSP and initial EPSP were expressed as percentage of the control values (MeanS.E.M.). Intravenous application of MK-801 remarkably attenuated the amplitude and duration of L-PSP. The strongest suppression was found at 15–20 min after administration (P<0.01, paired t-test).

same as that before ischemia (Fig. 3D). The amplitude of inhibitory postsynaptic potentials (IPSPs) was
2.813.05 mV (n=16) in CA1 neurons before ischemia. The IPSP was masked by L-PSP in L-PSP neurons. No significant difference in IPSP amplitude was detected between the control neurons and the Non L-PSP neurons at all time-points during reperfusion (
3.351.98 mV, n=32). The amplitude of IPSP in S-EPSP neurons was decreased to
0.470.82 mV at 31 h (n=7) and
0.901.56 mV at 42 h (n=3) after reperfusion but no statistical significance was detected. Paired-pulse facilitation The enhancement and depression of synaptic transmission induced by ischemia may be partly related to alterations of transmitter release and/or

postsynaptic response. Paired-pulse facilitation (PPF) is a short-lasting increase in synaptic efficacy and is considered to be presynaptic in origin.31,78 In hippocampus, when two stimuli are delivered to the input pathways in rapid succession, PPF manifests itself as an enhanced synaptic response to the second stimulus as it is delivered shortly after the first one. As shown in Fig. 4A, CA1 neurons before ischemia showed pronounced PPF of the slope and amplitude of initial EPSPs at 50 ms interpulse interval. After transient ischemia, no PPF was observed in L-PSP neurons while Non L-PSP and S-EPSP neurons exhibited obvious PPF. The temporal changes of PPF of these neurons following reperfusion were compared with EPSP paired-pulse facilitation index (EPI). The EPI was defined as the ratio of the slope of EPSP evoked by the second pulse to that of the

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Table 1. The size of late depolarizing postsynaptic potentials at different periods after reperfusion Reperfusion 5 h 19 h 31 h 42 h

Amplitude (mV)

Duration (ms)

n

5.062.75 5.393.79 4.882.68 3.192.38

93.3539.54 113.6047.51 72.1520.38* 83.9641.08†

12 23 24 10

Values are meansS.D. L-PSPs are elicited with 3T stimulus intensity. Duration of L-PSP is measured at half of its peak amplitude. n=cells/group. The number of neurons in this table is smaller than that in Fig. 1 because not all neurons go through this test. *P<0.01, †P<0.05 in comparison with 19 h (ANOVA). Part of these data were previously presented. More cells and time-points are added to this table.

first pulse.47 The EPI of L-PSP neurons was significantly smaller than that of pre-ischemic values (P<0.05 at 5 h, P<0.01 at 19–31 h). The EPI of Non L-PSP neurons increased at first 12 h after reperfusion and decreased thereafter. No statistical significance was detected from these changes. In S-EPSP neurons the EPI was the same as that of control levels (Fig. 4B). Localization of neurons with late depolarizing postsynaptic potential, non-late depolarizing postsynaptic potential and small excitatory postsynaptic potential The vulnerability to ischemia and the time-course of cell death are different even among CA1 pyramidal neurons. CA1 neurons in the medial portion are more sensitive to ischemia and degenerate faster than those in the lateral portion.14,60 It has also been suggested that calbindin-D28k (CaBP)-positive neurons may better tolerate excitotoxic insult50 and seizure activity65,70 than CaBP-negative neurons. In the CA1 pyramidal cell layer, the CaBP-positive neurons are in the superficial layer and show different responses to hypoxia in comparison with those in the deep layer.3,54 To examine the correlation between the postischemic responses of the neuron and its location, neurons with L-PSP, Non L-PSP or S-EPSP were divided into different subgroups according to their locations. As shown in Fig. 5A, the CA1 zone was divided into medial and lateral portions as well as deep and superficial layers. Approximately 70% of the neurons recorded in the deep layer were L-PSP neurons (57/83) while these neurons occupied 55% of the neurons recorded in the superficial layer (22/40, Fig. 5B). The pattern of localization of Non L-PSP neurons in the deep or superficial layer was reverse with 35% in the superficial layer (14/40) and 19% in the deep layer (16/83, Fig. 5B). However, no statistical significance was detected among such differences (÷2 =3.48, P=0.06). Among neurons recorded in the medial portion of CA1 zone, 76% were L-PSP neurons (41/54) and 11% were Non L-PSP neurons (6/54,

Fig. 5C). For neurons in the lateral portion, 55% were L-PSP neurons (38/69) and 35% were Non L-PSP neurons (24/69, Fig. 5C). These data indicated that more L-PSP neurons were located in the medial portion while more Non L-PSP neurons were in the lateral portion of CA1 zone (÷2 =9.22, P<0.01). No significant difference in localization was found in S-EPSP neurons. The proportion of each subgroup of neurons in the medial or lateral portion of CA1 zone at different intervals after reperfusion is presented in Fig. 5D. During the first 24 h of reperfusion, 80–90% of the neurons in the medial portion are L-PSP neurons. The percentage of L-PSP neurons reduced to 70% during 24–48 h after reperfusion while the S-EPSP neurons appeared at this time and occupied 10% of the population. In the lateral portion of hippocampus, L-PSP neurons occupied 50% of the population throughout the 48 h reperfusion period. DISCUSSION

The present study demonstrates that transient ischemia in vivo induces two distinct changes of synaptic transmission in CA1 pyramidal neurons. Synaptic transmission in the majority of the neurons was facilitated as evidenced by L-PSP and prolonged synaptic enhancement of initial EPSP. In contrast, synaptic transmission in a subpopulation of CA1 neurons was suppressed as evidenced by prolonged synaptic depression after ischemia. The enhancement or depression of synaptic transmission may play an important role in the pathophysiology of neuronal injury following transient forebrain ischemia. Prolonged synaptic enhancement in CA1 neurons following transient ischemia The facilitation of synaptic transmission induced by transient ischemia includes two components: the increase of amplitude and slope of initial EPSPs between 0–12 h and the development of L-PSPs between 4–48 h after reperfusion. This facilitation of synaptic transmission most likely represents enhanced synaptic efficacy because the excitability of these neurons progressively decreased following reperfusion.27 Similar changes have been observed in vitro76 and in vivo53 showing enhanced evoked field potentials in CA1 area while the excitability of CA1 neurons was decreased 5–10 h following an ischemic insult. The prolonged synaptic facilitation observed in the present study resembles anoxic long-term potentiation (LTP) induced in CA1 neurons in vitro by Cre´pel et al.16 Anoxic LTP is selectively mediated by NMDA receptors and requires a rise in intracellular Ca2+.15 In the present study, MK-801 significantly reduced the amplitude of initial EPSPs and L-PSPs suggesting that NMDA receptor-mediated activity may also be involved in maintenance of synaptic

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Fig. 3. Plots showing the changes of initial EPSP evoked from CA1 neurons by CC stimulation after ischemia. Each point is the mean value of percentage as compared with that before ischemia. (A) The slope of initial EPSP of neurons with L-PSP was significantly enhanced around 5 h after reperfusion (P<0.05, ANOVA) and gradually returned to pre-ischemic levels 31 h after reperfusion. In Non L-PSP neurons, however, the slope was persistently lower than pre-ischemic values up to 42 h following reperfusion (P<0.01). A remarkable reduction in the slope of initial EPSP was also observed in S-EPSP neurons (P<0.01). (B) The duration of the initial EPSP in L-PSP neurons gradually increased while that of Non L-PSP neurons reduced. (C) The stimulus threshold for inducing initial EPSP in both L-PSP neurons and Non L-PSP neurons gradually declined after reperfusion. (D) The latency of EPSP in all neurons was basically about the same as control value.

facilitation after ischemia in vivo. It is likely that the increase of glutamate concentration during ischemia combined with membrane potential depolarization during ischemic depolarization18,80 induces Ca2+ influx via ligand and voltage-regulated Ca2+ channels. Elevated intracellular Ca2+ may then trigger Ca2+-dependent facilitation of synaptic activity via phosphorylation of NMDA and AMPA receptors.36,63,66 These in turn may be responsible for the long-lasting changes in synaptic efficacy observed in this study. The change of PPF has been used as an indicator of presynaptic involvement in LTP generation.21,44,67 In the present study, PPF was reduced in L-PSP neurons suggesting the presynaptic involvement of synaptic facilitation after ischemia. A reduction of PPF has also been reported in CA1 region of hippocampal slice prepared from postischemic animals.46 It needs to be pointed out that the reduction of PPF in L-PSP neurons may be more apparent than real. In L-PSP neurons, the second EPSP is superimposed on the L-PSP induced by the first stimulation

(Fig. 4A), the slope and amplitude of the second EPSP may be reduced due to the following mechanisms: (i) electric shunting due to the increase of membrane conductance during L-PSP;25 (ii) the baseline from which the measurement was made is closer to the reversal potential of EPSPs. On the other hand, some studies have suggested the involvement of postsynaptic sites in the generation and maintenance of anoxic LTP16 and abnormal excitatory postsynaptic currents in CA1 neurons after ischemia.75 Results from the present study do not exclude the involvement of postsynaptic locus in the generation and maintenance of synaptic facilitation after ischemia. In fact, it is difficult to explain how ischemia could induce three different responses (i.e. L-PSP, Non L-PSP and S-EPSP) from CA1 neurons solely by presynaptic mechanisms. Previous studies have shown that CA1 pyramidal neurons in hippocampal slices prepared 28 h after ischemia exhibited prolonged synaptic responses, the later components of which were blocked by NMDA antagonists.35 In vivo extracellular recordings also

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Fig. 4. Ischemia-induced changes in the pair-pulse facilitation of CA1 neurons. (A) Representative pair-pulse responses in neurons before and after ischemia. Pair-pulse stimulation was delivered at interpulse interval of 50 ms. Each trace is the average of four individual sweeps. Pairpulse facilitation was present in control, Non L-PSP and S-EPSP neurons but was absent in L-PSP neurons. (B) Plots showing the temporal changes in the paired-pulse EPSP facilitation index (EPI), which was defined as the ratio of the slope of EPSP evoked by the second pulse to that of the first pulse following reperfusion. Each point is the mean value of EPI expressed as the percentage of the control one. The EPI in L-PSP neurons persistently declined to about 65% of the pre-ischemic levels (P<0.01, ANOVA). No significant changes in EPI were detected in Non L-PSP and S-EPSP neurons during the postischemic period.

demonstrated a NMDA receptor-dependent potentiation at Schaffer collateral/CA1 pyramidal cell synapses beginning at 6–8 h following transient cerebral ischemia.53 The timing and characteristics of the electrophysiological changes in the above studies closely resemble the L-PSPs observed in the present study. The L-PSPs induced by transient ischemia in this study persisted up to two days following reperfusion and were greatly attenuated by the NMDA channel blocker MK-801. Although pharmacologic doses of MK-801 were shown to be ineffective in protecting CA1 neurons in the four-vessel occlusion rat model when given during the first 16 h,8 it remains possible that NMDA-related mechanisms participate in neuronal degeneration at later times. L-PSPs may play an important role in pathogenesis of postischemic cell death by enhancing Ca2+ influx through membrane potential depolarization and

activation of NMDA components. The possible mechanisms underlying the facilitation of NMDA components after ischemia include the reduction of Mg2+ blockade of NMDA receptors;34 modulation of redox sites in the receptors29 or attenuation of synaptic inhibition.51,77,81 Moreover, a similar NMDA receptor-mediated late synaptic response has been recorded in the CA1 region of hippocampal slices after blockade of GABAA receptors17 or adenosine A1 receptors43 in normal condition or after an anoxic-aglycemic episode,15,74 suggesting that a NMDA receptor-mediated local excitatory network19,73 may be unmasked after ischemic insult. In the present study MK-801 only blocks 60% of L-PSPs. One possibility is that the dose of MK-801 in the present study may be insufficient for a complete blockade of NMDA receptors. The other possibility is that the remaining component of L-PSPs might be mediated by AMPA receptors. Under physiological conditions, EPSPs mediated by AMPA receptors have faster rising and decay times than those mediated by NMDA receptors.40 AMPA receptors are usually impermeable to Ca2+.39 However, it has been shown in hippocampal slice that CA1 neurons displayed unusually slow excitatory postsynaptic currents by activation of AMPA receptors after ischemia.75 This AMPA-activated current also mediated Ca2+ influx. Several in vivo studies suggest that the Ca2+ influx through selective AMPA receptor coupled channels during reperfusion may be one of the mechanisms of postischemic cell death. For example, the AMPA receptor antagonist 3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) completely blocked the increase of Ca2+ influx in CA1 neurons by stimulation of perforant pathway 6 h after reperfusion;1 AMPA receptor antagonists given after ischemia showed protective effects on CA1 neurons.7,56,68 A potential mechanism for postischemic increase in Ca2+ permeability through channels linked to AMPA receptors is the selective reduction in expression of Glu2 receptor subunits after ischemia.28,58 Prolonged synaptic depression in CA1 neurons following transient ischemia In contrast to L-PSP neurons, Non L-PSP neurons failed to develop L-PSPs but displayed a prolonged depression of synaptic transmission following reperfusion. To our knowledge, this is the first report showing that the synaptic transmission in some CA1 pyramidal neurons is persistently suppressed up to two days following reperfusion. This ischemiainduced synaptic depression resembles the long-term depression (LTD) that has been recently described and widely studied in the CA1 region of hippocampus in vitro5,20,55 and in vivo.33 It has been suggested that large Ca2+ influx leads to LTP via activating Ca2+-dependent protein kinases while modest Ca2+ influx results in LTD via selectively

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Fig. 5. The localization of CA1 neurons with three distinct responses after ischemia. (A) A schematic drawing illustrates how the location of a pyramidal neuron was determined in the present study. The peak of the natural curve of CA1 pyramidal cell layer was used to divide medial and lateral portion of CA1 zone. The neurons in the dorsal tier of CA1 pyramidal cell layer were considered as the deep layer neurons and those in the ventral tier were considered as the superficial layer neurons. (B) Histogram showing the distribution of recorded neurons in the deep layer or superficial layer of CA1 zone. Among neurons recorded in the deep layer, 70% were L-PSP neurons and 19% were Non L-PSP neurons. For neurons in the superficial layer, 55% were L-PSP neurons and 35% were Non L-PSP neurons. The distribution of S-EPSP neurons in the deep layer or superficial layer was about the same. (C) Histogram showing the localization of neurons in the medial or lateral portion of CA1 zone. Approximately 75% of the recorded neurons in the medial portion were L-PSP neurons while only 10% were Non L-PSP neurons. On the other hand, 55% of neurons in the lateral portion were L-PSP neurons while 35% were Non L-PSP neurons. These data indicated that more L-PSP neurons were located in the medial portion and more Non L-PSP neurons were in the lateral portion (÷2 =9.22, P<0.01). No significant difference in localization was found in S-EPSP neurons. (D) Histogram showing the proportion of CA1 neurons with different synaptic responses in the medial or lateral portion at different intervals after reperfusion. In the medial portion, 80% of recorded neurons were L-PSP neurons around 5 h after reperfusion and then increased to 90% at around 19 h after reperfusion. The proportion of L-PSP neurons declined to 70% during 31–42 h after reperfusion while S-EPSP neurons occupied 10–20% of the population during this period. Non L-PSP neurons only accounted for less than 20% of the neurons in the medial portion. In the lateral portion, the proportion of L-PSP neurons and Non L-PSP neurons was about the same (40–50%) during the whole reperfusion period.

activating protein phosphates.2,48,49 Changes in the amplitude of fast afterhyperpolarization (fAHP) may serve as indirect evidence of intracellular Ca2+ rise. fAHP is mediated by calcium-activated potassium currents and the amplitude of fAHP is closely related to the intracellular Ca2+ concentration.45,71 Consistent with the above hypothesis, the amplitude of fAHP in Non L-PSP neurons is smaller than that of L-PSP neurons,27 suggesting a smaller rise of intracellular Ca2+ in Non L-PSP neurons than in L-PSP neurons after ischemia. The majority of Non L-PSP neurons are located in the lateral portion of hippocampus (Fig. 5C), where neurons are less vulnerable

to ischemia than those in the medial portion.60 Non L-PSP neurons may be the neurons recovering from ischemic insult. The long-lasting synaptic depression after ischemia may represent a cell property that increases the ischemic resistance of these neurons. The depression of synaptic transmission was also observed in S-EPSP neurons but was quite different from that in Non L-PSP neurons. S-EPSP neurons appeared 31 h after reperfusion with great depression of synaptic transmission while the depression of Non L-PSP neurons started to recover at this time (Fig. 3A). In addition, the membrane properties of S-EPSP neurons progressively deteriorated while the

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changes of membrane properties in Non L-PSP neurons was transient.27 Therefore, the depression of synaptic transmission in S-EPSP neurons may be a sign of cell degeneration (see discussion below). Neurophysiological changes and neuronal damage following ischemia It has been shown that most of the CA1 neurons die one week after 13 min ischemic depolarization in this model.62 After ischemia of the same severity in the present study, pyramidal neurons in the medial portion of CA1 zone begin to show histological signs of cell death at 37–48 h after reperfusion. L-PSP neurons were 80% of the recorded neurons in the medial portion 5 h after ischemia and increased to 90% at 19 h after reperfusion. The number of L-PSP neurons were reduced to 70% at 31 h and 42 h after reperfusion but at this time 10% of the neurons showed S-EPSPs (Fig. 5D). In contrast, more Non L-PSP neurons were in the lateral portion (40–50%) than in the medial portion (10–20%). The membrane properties of L-PSP neurons and S-EPSP neurons progressively deteriorated after reperfusion, whereas those of Non L-PSP neurons were transient27 and resembled what has been observed in CA1 neurons after 5 min ischemia.81 Based on the above observations, it is conceivable that L-PSP neurons and S-EPSP neurons are the degenerating neurons and the Non L-PSP neurons are the surviving neurons after ischemic insult. Neurons with L-PSP may represent the early phase of postischemic cell damage while neurons with S-EPSP may be in the late phase of cell degeneration. It has been shown that following transient increase in intracellular Ca2+ during ischemia, a second rise in intracellular Ca2+ developed in 50–60% of the CA1 neurons 2–8 h after reperfusion.69 The proportion of CA1 neurons with secondary increase in intracellular Ca2+ is very close to that of L-PSP neurons observed in the present study. If this delayed increase of intracellular Ca2+ concentration is an important component of postischemic neuronal death, the Ca2+ buffering capacity may be an important determinant in the survivability of the neurons following ischemia. However, it has been reported that no relationship

between calcium binding protein content and ischemic injury was found in hippocampus and other forebrain structures.22 Other studies, in contrast, indicated that hippocampal neurons that do not express calcium-binding protein, such as CaBP or parvalbumin, have higher sensitivity to excitotoxicity than those that do.50,70 It has been shown that most of the neurons in the deep layer of CA1 zone are CaBP negative and have different responses to hypoxia than those in the superficial layer which are CaBP positive.3,54 More L-PSP neurons are located in the deep layer of CA1 zone (Fig. 5B) and probably are CaBP-negative neurons, which makes them highly susceptible to ischemia. On the other hand, more Non L-PSP neurons are in the superficial layer (Fig. 5B) and presumably are CaBP positive, which may help to resist ischemic insult. Despite the evidence in the present study indicating that Non L-PSP neurons are the surviving neurons after ischemia, the overall proportion of these neurons (30%) is higher than expected. One explanation is that it is always easier to record from a ‘‘healthy’’ neuron than from a degenerating neuron, so the proportion of Non L-PSP neurons (presumably surviving neurons) is most likely over represented in the present study. On the other hand, neurons with Non L-PSP may represent a different type of cell degeneration following ischemia. It has been suggested that two different pathways exist in postischemic cell death: the neurons with higher intracellular Ca2+ die fast and may be associated with necrotic cell death while the neurons with lower intracellular Ca2+ may undergo the apoptotic cell death.11 The amplitude of fAHP in Non L-PSP neurons is smaller than that of L-PSP neurons but larger than that of control ones27 suggesting that a moderate Ca2+ increase does occur in these neurons. Non L-PSP neurons may succumb to late developing apoptotic mechanism. Acknowledgements—We thank Drs S. Hestrin and T. Nowak for critical reading and helpful comments on this manuscript. This research was supported by grants AHA 94008130, NIH NS33101 to Z.C.X. and Semmes-Murphey Chair of Excellence to W.A.P. T.M.G. is the recipient of grants NNSF 39570263 and NSFGP 950537 of People’s Republic of China.

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