The NMDA antagonist, MK-801, suppresses long-term potentiation, kindling, and kindling-induced potentiation in the perforant path of the unanesthetized rat

89

Brain Research, 519 (1990) 89-96 Elsevier

BRES 15552

The NMDA antagonist, MK-801, suppresses long-term potentiation, kindling, and kindling-induced potentiation in the perforant path of the unanesthetized rat* M.E. Gilbert and C.M. Mack NSI Technology Services Corporation, Environmental Sciences, Research Triangle Park, NC 27711 (U.S.A.)

(Accepted 28 November 1989) Key words: Kindling; Perforant path; Long-term potentiation; N-Methyl-o-aspartate; MK-801

Antagonism of NMDA-mediated transmission by MK-801 has been shown to block long-term potentiation (LTP) in vitro and delay electrical kindling of the amygdala. The present experiment sought to examine the relationship between synaptic potentiation of the perforant path-granule cell synapse and development of perforant path kindling. MK-801 (0.1 and 1.0 mg/kg) blocked induction of LTP of the perforant path in the unanesthetized animal measured 24 h after train delivery. The 1.0 mg/kg dosage also increased afterdischarge (AD) thresholds, delayed kindling development from daily stimulation of the perforant path (,~ = 8.82 + 1.19 and 22.9 + 3.66 sessions to the first stage 5 seizure), and increased AD durations. Kindling produced a significant potentiation of the EPSP (47%) and population spike (49%) after the first evoked AD in control animals. No significant enhancement of either component of the field potential was observed in MK-801-treated animals. Animals treated with this dosage of MK-801, did, however, kindle in the absence of potentiation at this synapse. It was concluded that although NMDA-mediated potentiation may facilitate kindling, synaptic potentiation does not appear to be a critical requirement for kindling to develop. These findings support the notion that development of the burst response and not synaptic enhancement may be the critical physiological alteration that underlies the kindling phenomenon. INTRODUCTION Kindling is a model of temporal lobe epilepsy in which seizures are gradually induced by daily electrical stimulation of a variety of brain sites 11. Although the mechanisms underlying the permanent change in brain function that subserves kindling remain elusive, it is generally accepted that kindling promotes a net increase in transmission along pathways activated by the kindling stimulus 43. Long-term potentiation (LTP) refers to a long-lasting increase in synaptic efficacy following the administration of high-frequency, non-epileptiform-inducing trains of stimulation 2. The enhancement of synaptic transmission is most dramatic and of longest duration in the hippocampus 34. Kindling also produces potentiation in the hippocampus 7'8'23'43 and repeated administration of LTP trains can facilitate subsequent kindling 36'44. This has led several investigators to propose that the increase in synaptic efficacy induced by kindling may contribute to the development of a seizuregenic

focus and spread of epileptogenesis through an LTP mechanism 6'16'24. Others have argued that the two p h e n o m e n a are distinct - - they do not rely on similar mechanisms as several temporal, electrographic, site specific and pharmacological distinctions exist between them3,31,32,42,43. One commonality in the two phenomena, however, is the recent demonstrations of their disruption by agonists and antagonists of the glutamate receptor subtype, N-methyl-D-aspartate ( N M D A ) . N M D A can induce both LTP 15'2°'45 and seizures activity 29"46. Antagonists of the N M D A receptor (APV, CPP, MK-801) block the induction of hippocampal LTP in CA1 in vitro 5'6"14 and the dentate gyrus of anesthetized preparations ~. N M D A antagonists (APV, MK-801) have also been shown recently to delay profoundly the development of amygdala kindling 4'9'25'39, and suppress kindled seizures 9'25'48. Some of these reports have emphasized the importance of N M D A receptor activation in LTP and suggested the contributory role of such an increase in synaptic strength

* Although the research described in this article has been supported by the United States Environmental Protection Agency (through contract 68-02-4450 to NSI Technology Services Corp.), it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Correspondence: M.E. Gilbert, Neurotoxicology Division, MD 74B, Health Effects Research Laboratories, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, U.S.A. 0006-8993/90/$03.50 I~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

90 to the kindling p r o c e s s 9'25"39. The present series of experiments was designed to study further the relationship between LTP and kindling through pharmacological manipulations of NMDA-mediated transmission. MK-801 is a potent non-competitive NMDA antagonist which produces a block of the calcium and sodium conductance through voltage-gated channels opened by glutamate occupation of the NMDA receptor ~2. The effects of MK-801 on LTP of the dentate gyrus from train delivery to the perforant path were evaluated in chronically implanted, non-anesthetized animals. Although several instances of LTP disruption by NMDA antagonists have been demonstrated in area CA1 of the hippocampus in vitro6, few have documented the phenomenon in the dentate gyrus in vivo (but see Abraham and Mason1), and no reports exist for the unanesthetized preparation. Once we had established MK-801's action on LTP, we sought to study its effects on kindling of the same site, the perforant path. Previous investigations have utilized the amygdala as the kindling site 9'25'39. Finally, the coincident potentiation associated with a kindling stimulation and/or subsequent afterdischarge (AD) was monitored throughout kindling of the perforant path in animals treated with MK-801.

MATERIALS AND METHODS

Subjects Adult male Long-Evans rats from the Charles River Breeding Company served as subjects. Animals were individually housed in plastic cages with woodchip bedding in a colony room maintained on a 12:12 h light:dark schedule and permitted free access to food and water at all times. At 90-120 days of age, animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and administered atropine sulphate (0.2 mg/kg, i.p.). Bipolar electrodes were implanted in the angular bundle of the perforant path and the hilus of the dentate gyrus. Exact electrode placement was achieved using electrophysiological monitoring and according to standard stereotaxic procedures described previously1°. Nominally, stereotaxic coordinates with the skull surface flat were 7.8 mm posterior to bregma, 4.4 mm lateral to the midline, and approximately 2.2 mm ventral to dura for the perforant path, and 3.5 mm posterior to bregma, 2.2 mm lateral to the midline, and 3.5 mm ventral to dura for the dentate gyrus. Ten additional animals with electrodes in the perforant path alone were prepared for kindling purposes. Electrodes were constructed of stainless-steel wire (each strand 125/~m in diameter), insulated with teflon except for the cut tips, and crimped onto gold-plated Amphenol pins. The tips of the electrodes were separated vertically by 0.5 mm. The Amphenol pins were inserted into a 9-pin connector, which was cemented to the animal's skull and anchored with stainless steel screws (23 × 1.6 mm). The animals were grounded through a screw inserted in the skull overlying the anterior neocortex. Immediately following surgery all animals received an i.m. injection of penicillin G (100,000 units).

Input~output functions A minimum of two weeks following surgery, animals with electrodes in the perforant path and dentate gyrus were connected to a Pathfinder II (Nicolet Biomedical, Madison, WI) via a lownoise shielded cable and administered biphasic square wave stimulus pulses (each phase 0.1 ms in duration) using a Grass S-88 stimulator

and PSIU-6 constant current converters. The response evoked in the dentate gyrus to pulses applied to the perforant path was amplified and averaged over 10 trials. Ten pulses were delivered at each of 5 stimulus intensities ranging from 20 to 1200/~A and adjusted for each animal to span the range from subthreshold for population spike generation, to asymptotic levels. Two measures, population spike height and excitatory postsynaptic potential (EPSP) slope, were taken from each averaged field potential. Height of the population spike was calculated by taking the average of the two peak-to-peak amplitudes comprising the negative potential, and constitutes a measure of the number of granule cells firing21. The slope of the rising phase of the EPSP is a measure of the strength of synaptic response~. This was assessed by measuring the slope of the line from a fixed latency after delivery of the stimulus pulse (approximately 1.5 ms) to the peak of the positive potential or onset of the population spike. Daily input/output (I/O) functions were collected until three consecutive days of stable response curves were obtained. Animals were placed in the testing box for a minimum of 10 min prior to data collection. Only animals whose responses were stable and whose maximum evoked potential was a minimum of 3 mv were included in the LTP and I/O analysis phase of the kindling experiment.

Long-term potentiation I/O functions were obtained prior to administration of 1.0 ml/kg of saline (n = 9), 0.1 mg/kg (n = 7) or 1.0 mg/kg (n = 8) of MK-801. The high dosage produced behavioral signs of toxicity including circling and nodding. The low dosage was without overt behavioral effects. A second I/O function was generated 20 min later and was followed by administration of 5 sets (2 s apart) of two high frequency trains (50 ms, 400 Hz, 0.1 ms pulse duration, 1000/zA) at 10 min intervals. This intensity produced nearly asymptotic field potentials with a single pulse. Two more I/Os were collected 15 min and 24 h after the last set of trains. If field potentials remained stable upon decay of LTP (3-4 weeks post-train delivery), animals were administered a second set of LTP trains under a different drug condition. Following a second decay period, animals were kindled in the perforant path.

Kindling Two weeks following surgery, or 3-4 weeks following LTP, animals were individually placed in a Plexiglas chamber and connected to the stimulating apparatus. The EEG was recorded on a Grass polygraph before and after stimulation to record a prestimulation baseline and evoked AD. An AD was defined as rhythmic spiking, at least 4 times the amplitude of the baseline EEG, sustained for a minimum of 5 s. AD was monitored from the perforant path and dentate gyrus by recording between the tips of the bipolar electrode. For AD threshold testing, stimulation consisted of a 1 s train of 60 Hz biphasic squarewaves, each 1.0 ms in duration. AD threshold testing began 30 min following administration of 0 (n = 14) or 1.0 mg/kg (n = 23) of MK-801. Thresholds were determined by administering an ascending series of stimulations to the perforant path, beginning at 100 ~A and increasing in 100/~A steps, at 1 min intervals, until an AD was observed. Some animals treated with MK-801 were found to have thresholds in excess of 1000/~A. In this event, AD threshold testing was resumed the following day but utilizing a 2 s stimulus train. For purposes of analysis, these animals were assigned 1000 /~A as threshold. Following threshold determination, stimulation was delivered once daily at a standard 800/tA 2 s train until 3 stage 5 seizures3° were observed.

Afterdischarge-induced potentiation A subset of the animals in the kindling phase of the experiment were monitored for stable field potentials as outlined above. Some of these animals were used in the LTP phase of the experiment (n = 7, see above) and the remainder were naive (n = 18). I/O functions were taken prior to drug administration and AD threshold testing. I/Os were monitored 18-24 h after the 1st, 4th, 7th, 10th,

91 and 13th AD, after the first signs of generalized seizure activity (stages 3-4), and after at least one stage 5 seizure. Three animals in the dosed group whose AD thresholds were in excess of 1000/~A using a 1 s kindling stimulation train were tested undrugged the following day. No potentiation of the I/O function was observed following the first threshold test in the absence of an AD. A D was evoked and field potentials were enhanced in these animals following the second threshold test in the absence of a drug. The I/O data from the second threshold test were included in the control group for determination of potentiation induced after the 1st A D as this was obtained in the undrugged state. Thereafter, these animals were stimulated 30 rain following MK-801 administration and were excluded from the kindling-induced potentiation analysis. They were included, however, in analysis of kindling development.

Data transformation and statistics Since the amplitude of an individual subject's evoked response varied with respect to other animals within a given treatment group, I/O data were normalized. This was achieved by expressing the EPSP and population spike height measurements of each subject as a percentage of the maximum response recorded for that animal at baseline. The 4 I/O functions collected during the LTP run were compared using repeated measures ANOVA. Evidence of LTP was determined by assessing each of the three dosage groups separately. Step-down ANOVAs were used to evaluate between timepoint comparisons (pre-drug/post-drug, post-drug/post-train, and predrug/24 h post-train) in the event of a significant main effect of time. Kindling-induced changes in I/O functions for each condition were assessed statistically by comparing prekindling baseline I/Os with the I/O collected 24 h after the 1st and 10th evoked A D using repeated measures ANOVA. Sessions in each stage of motor seizure and AD development over the first 8 ADs were also subjected to repeated measure ANOVA. AD threshold and mean AD duration associated with Stage 5 seizures were subjected to one-way ANOVAs. Pearson-product moment correlation coefficients were calculated to assess the relationship between the degree of synaptic potentiation and kindling rate. The percent change from baseline of the population spike and the EPSP slope following the first AD (at an intensity producing a response 80% of the baseline maximum) and the number of sessions to reach the first stage 4 seizure were analyzed for treated and control subjects (n = 15).

0.70) (see left panel of Fig. 1B). Unlike the population spike, no potentiation of the EPSP was evident in control animals (F3,24 = 0.26, P > 0.85) (right panel of Fig. 1A). A significant effect of hour in the 1.0 mg/kg (/'3,21 = 4.19, P < 0.0179) dosage group is due to an MK-801-induced decrease in EPSP slope. A step-down ANOVA test revealed a significant reduction in the slope of the EPSP post-drug relative to baseline (F1,7 = 6.44, P < 0.0388). Twenty-four h following train delivery, EPSP slope had returned to baseline levels (P > 0.95) (right panel Fig. 1C).

Kindling A total of 14 animals were dropped from the main analysis of the study - - 3 control and 6 treated animals lost headplugs, and 1 treated animal died prior to the completion of kindling. An additional 4 treated animals failed to kindle despite 100 stimulation sessions. Data for AD thresholds and AD development were included for some of these subjects as described below. MK-801 significantly increased AD thresholds (~ =

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Seizure Stage Fig. 2. The effects of MK-801 on perforant path kindling. A: mean (+ S.E.M.) afterdischarge (AD) duration over the first 8 stimulation sessions in controls and animals treated with 1.0 mg/kg of MK-801. B: mean (+ S.E.M.) number of sessions in each stage of motor seizure for control and MK-801-treated animals. Results of ANOVA for dose and dose x session (D × Sess) or Dose × Stage (D x Stg) interactions are indicated.

721.74 + 62.86) relative to controls (2 = 500.0 _ 28.01; }71,39 = 8.65, P < 0.0056). A 1-s train duration failed to evoke an AD in 11/23 animals treated with 1.0 mg/kg MK-801, so a maximum of 1000/tA was assigned for analysis purposes. A 1-s train duration was effective in evoking AD in all animals tested under saline conditions (n = 14). Four animals treated with MK-801 who had failed to experience an A D on the initial threshold testing day (3 whose evoked responses were monitored, and a fourth animal who possessed only a perforant path kindling electrode), were given a second threshold test the following day in the absence of drug. ADs were observed in all four of these subjects between 400 and 800 /~A. Thresholds for these animals under both drugged and undrugged conditions were included in the statistical analysis reported above. The 2-s 800/~A stimulus train used in subsequent sessions evoked AD in all treated and control subjects. Despite higher thresholds in treated animals, the development of A D over the first 8 ADs was enhanced by MK-801. Fig. 2A presents the mean AD duration

recorded from the perforant path electrode of control and treated animals. A significant effect of dose in the absence of a dose × session interaction (see Fig. 2A for results of statistical tests) indicates that this prolonged AD duration in the treated animals occurred acrosss all sessions. MK-801 delayed the development of perforant path kindling from a control mean of 8.82 + 1.19 (n = 11) evoked ADs to the first Stage 5 seizure to 22.92 + 3.66 (n = 12). A total of 10 animals (3 control, 7 treated) dislodged their headplugs prior to completion of kindling and were not included in the computation of this mean. Four additional MK-801-treated animals failed to reach Stage 5 seizures after 65 stimulation sessions. Stimulation was increased to 2/day until 100 ADs had been evoked. These subjects had still failed to progress beyond Stage 2 seizures. After a 48-h drug-flee period, these animals were stimulated once daily in the absence of MK-801 and Stage 5 seizures were observed within 6-13 sessions. The mean of 9.3 sessions was similar to control mean of 8.82, suggesting very little savings in kindling development under the no-drug condition, despite the prolonged stimulation history and evoked ADs. A summary of the number of sessions in each stage of motor seizure is presented in Fig. 2B. This depiction and analysis excludes all treated (n = 11) and control (n = 3) animals described above who had not experienced at least 1 Stage 5 seizure, but clearly demonstrates that the delay in kindling development induced by MK-801 is restricted to Stage 1, when the seizure is assumed to be focal in nature. A delay in the progression from Stage 4 to Stage 5 is also apparent. This may not be so much a failure of the seizure to fully generalize as it is a reflection of the ataxic state of the treated animals. Stage 5 seizures, operationally defined, entail rearing with bilateral forelimb clonus and loss of postural control 3°. Most animals treated with 1.0 mg/kg of MK-801 were severely impaired motorically. Bilateral clonus was readily observed (Stage 4), but the inability to rear promoted difficulty in defining the occurrence of Stage 5 seizures. Interestingly, the duration of clonic seizure activity associated with Stage 5 seizures did not differ (,~ = 24.58 + 1.76 and 26.92 + 1.92 s, for control and treated groups, respectively), despite a prolonged focal A D associated with this stage of kindling in treated animals (32.18 + 3.25 vs 58.92 + 5.28; F1,21 = 17.77, P < 0.0005).

Kindling-induced potentiation Kindling-induced potentiation of the population spike and EPSP slope are presented in the left hand columns of Fig. 3A,B. The first A D produced near maximum enhancement of the population spike and EPSP slope that was most prominent at the higher stimulus intensities

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Fig. 3. (A) Mean (+ S.E.M.) EPSP slope and (B) population spike amplitudes (+ S.E.M.) in control (left panel) and MK-801-treated (right panel) animals. Prekindling baseline I/Os are contrasted with I/O functions taken 24 h after the 1st (AD1), 4th (AD4), 7th (AD7), 10th (AD10), and 13th (AD13) evoked ADs. Population spike height and EPSP slope are expressed as percentages of the maximum response recorded for each animal at baseline. A statistically significant increase in EPSP and population spike height was seen in control animals at AD1 and AD10. No such potentiation in either component was evident in animals treated with 1.0 mg/kg MK-801 throughout kindling (see text).

(intensity x session interaction F4,44 = 2.77, P < 0.0386 for population spike and F4.44 = 4.18, P < 0.0059 for E P S P slope) in control animals. In contrast, no A D induced potentiation of the population spike (F1,12 = 0.21, P > 0.65) or EPSP (F1,12 = 2.74, P > 0.12) was observed in MK-801 treated animals following the first A D . Neither was there evidence for a significant intensity x session interaction for either measure (both P's > 0.26). Slight increases in population spike occurred at the highest intensities after 4-13 A D s , but were still of smaller magnitude than that observed in controls (Fig. 3A). The E P S P failed to demonstrate any increases over the 13 A D s for which it was monitored, and in fact slight decrements in EPSP slope were observed. A repeated

measures A N O V A of I/O functions following the 10th A D confirmed that increases in population spike height (F4,16 = 6.44, P < 0.0027) and E P S P slope (F4,16 = 3.11, P < 0.0451) were maintained throughout kindling in control animals. Changes in I/O functions in MK801-treated animals at similar time points failed to reveal significant fluctuations from baseline for either measure and no evidence of significant intensity x session interactions (all P's > 0.1). Neither was there evidence of increases in the EPSP slopes or population spikes of treated animals in I/Os taken after the first signs of seizure generalization (Stages 3-4) (Fig. 4) or the occurrence of at least one stage 5 seizure (data not shown).

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An examination of data from individual animals failed to reveal a relationship between the amount of potentiation of the population spike (r = -0.10) or EPSP slope (r = -0.17) and kindling rate. DISCUSSION MK-801 reduced the slope of the EPSP, blocked LTP of the perforant path to dentate gyrus synapse in the unanesthetized animal, delayed the development of perforant path kindling, reduced kindling-induced potentiation of the population spike, and completely blocked kindling-induced potentiation of the EPSP. Each of these effects will be discussed separately below.

Long-term potentiation MK-801 produced a dose-related decrease in the slope of the EPSP prior to the induction of LTP trains. This finding was unexpected given that N M D A antagonists are generally reported to have no effects on low frequency, single pulse evoked potentials in vitro 22. They do, however, confirm recent in vivo findings by Abraham and Mason 1 who also report an MK-801-induced decrease in EPSP slope at the perforant path-granule cell synapse of the anesthetized rat. MK-801's blocking effects on LTP were evident at a dosage that failed to produce overt behavioral signs of drug intoxication. This is the first report of NMDAreceptor antagonists effects on LTP in the unanesthetized preparation and may be of significance in the evaluation of MK-801 disruption of learning and memory 38'47.

It is curious that no increase in EPSP slope was observed in control animals following delivery of LTP trains. The population spike is typically used as the measure of LTP in chronic studies as it demonstrates a larger magnitude of increase and is more stable from recording to recording. In vitro or in vivo studies using anesthetized preparations report large and stable potentiation of the EPSP, but chronic studies are plagued with high variability of this measure 34. Since movement can affect the size of hippocampal evoked potentials 33, an attempt was made to record from animals while they were in a quiet, resting state. The absence of potentiation of the EPSP may be related to variability of the measure and uncontrolled fluctuations in the behavioral state of the animal.

Kindling Several laboratories have now demonstrated a retardation of amygdala kindling development with daily treatment with MK-8019'25'39. The present findings extend the effects of this N M D A antagonist to the site most frequently investigated in chronic LTP studies. However, several differences between amygdala 9 and perforant path (present study) kindling sites were observed. MK801 increased A D thresholds in the perforant path, an effect we had not previously seen in amygdala 9. In the latter study, however, a lower dosage of MK-801 was used (0.5 as compared to 1.0 mg/kg). In fully kindled animals MK-801 resulted in a selective increase in threshold in dorsal hippocampus as compared to amygdala. Morimoto et al. 26'27 have proposed that the balance between GABA-mediated inhibition and NMDA-mediated excitation is the critical mechanism for the initiation of an AD. In the hippocampus strong GABA-mediated recurrent inhibition suppresses the E E G during the delivery of the tetanic train. When the stimulus is terminated, rhythmic discharge is spontaneously generated and precedes the initiation of the AD. With long stimulus trains (s) the inhibition fails and results in an unleashing of NMDA-mediated excitation and the promotion of an AD 27. The N M D A antagonist, APV, increases the duration of the suppression during stimulation, and delays the onset of the rhythmic synchronous discharge 27. NMDA-antagonism by MK-801 in the present study may have increased seizure thresholds by a similar mechanism. In the amygdala focus, once an A D had been triggered, MK-801 shortened its duration over the first 10 sessions of kindling 9. In the present study, MK-801 increased A D durations recorded from the perforant path stimulating electrode over the first 8 sessions. An increase in A D duration was revealed coincident with a

95 delay in kindling development. Longer perforant path ADs were also recorded in the presence of unaltered motor seizure durations (duration of clonus associated with Stage 5 seizures). Kairiss et al. 18 have reported that LTP trains delivered to the perforant path potentiate both excitatory and feed-forward inhibitory circuits in the dentate gyrus. Since MK-801 is effective in blocking potentiation of excitatory synaptic transmission (following LTP- and AD-inducing trains, see below), it may also block potentiation of feed-forward inhibition. This type of inhibition may contribute to the termination of an AD. Kindling-induced potentiation In agreement with previous reports 7'8"23'34'43, potentiation of the EPSP and population spike were evident after the first kindling stimulation. MK-801 completely blocked AD-induced potentiation of the EPSP and suppressed the degree of potentiation evident in the population spike. Neither was potentiation of this synapse observed with the onset of seizure generalization, when certainly areas beyond the primary kindled site are contributing to the seizure. The seizure has propagated beyond its focal triggering site to incorporate forebrain and/or brainstem areas responsible for driving the motor components of the generalized seizure. This occurred in the absence of potentiation of an excitatory synapse directly activated by stimulus train and evoked AD. Furthermore, there appears to be little correspondence between the amount of potentiation and the rate of kindling development. Animals demonstrating dramatic enhancement of the population spike or EPSP did not necessarily kindle faster than those showing very little potentiation, regardless of dose group. Conversely, modest potentiation, or its absence, was not a reliable predictor of a delay in kindling development. These data suggest that synaptic potentiation of the primary evoked potential is neither necessary nor sufficient for generalized seizures and/or kindling to occur. Such a conclusion is consistent with previous findings demonstrating that the integrity of the dentate granule cells or the hippocampus proper is not required for the induction or maintenance of entorhinal cortex, septal or amygdala

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Acknowledgements. The authors would like to acknowledge the technical assistance of Shawn Acheson and Christina Murchison, and are grateful to Drs. Karl Jensen and Mark Stanton for their critiques of an earlier version of this manuscript. Numerous invaluable discussions with Dr. Linda Burdette are also gratefully acknowledged. The MK-801 was a generous gift from Merck, Sharp and Dohme.

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