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Glutamate Cascade Research

The Glutamate Cascade: Common Pathways of Central Nervous System Disease States

Poster Abstracts


GLUTAMATE-DOPAMINE INTERACTIONS IN HUMANS (GII-B)
Abi-Saab, W.M.; D'Souza, D.C.; Karper, L.P.; Madonick, S.; Belger, A.; Gil, R.; Bennett, A.; Abi-Dargham, A.; Heninger, G.R.; Charney, D.S.; and Krystal, J.H.

The interactions of glutamate and dopamine systems have been implicated in the pathophysiology of several neuropsychiatric and substance abuse disorders. To study these interactions, we are examining the effects of dopamine agonists and antagonists on subhypnotic doses of the N-methyl D-aspartate (NMDA) glutamate receptor antagonist, ketamine, in humans.

METHODS: The two studies employed a randomly balanced, placebo-controlled design where in the first study healthy subjects (n=20) received haloperidol 5 mg or placebo 2 hours prior to ketamine (0.26 mg/kg bolus then 0.65 mg/kg over 1 hour) or placebo; or in the second study, healthy subjects (n=7) received amphetamine (0.25 mg/kg over 1 min) or placebo and ketamine (0.23 mg/kg bolus then 0.5 mg/kg over 1 hour) or placebo.

RESULTS: Study 1: Ketamine produced psychosis, perceptual alterations, and performance decrements in abstraction and Wisconsin Card Sorting Test (WCST). While haloperidol did not alter psychosis, perceptual changes, or euphoria produced by ketamine, it was effective in reducing the abstraction and WCST impairments. Study 2: Our preliminary data suggest that amphetamine had no effect on the ketamine-induced psychosis or euphoria. However, it may have reduced attention deficits produced by ketamine.

IMPLICATIONS: First, the impairments in executive cognitive functions arising from deficits in NMDA receptor function appear to be mediated in part by excessive stimulation of dopamine-2 receptors. Second, neither the psychosis nor the euphoria produced by NMDA antagonists appear dependent upon D2 receptor stimulation. Third, the failure of amphetamine to exacerbate the ketamine psychosis may suggest that the NMDA receptor deficit model is most relevant to a subset of schizophrenic patients with poor responses to typical neuroleptics, since neuroleptic responsive patients tend to show worsening of psychosis during amphetamine infusion.


LAMOTRIGINE EFFECT ON KETAMINE INDUCED NEUROPSYCHIATRIC SYMPTOMS IN HUMANS (GII-B)
Anand, A.; Charney, D.S.; Cappiello, A.; Berman, R.M.; Oren, D.A.; and Krystal, J.H.

Ketamine is a non-competitive antagonist of the NMDA receptor that produces psychosis, perceptual alterations, thought disorder, and mood changes in healthy subjects. Preclinical studies suggest that ketamine may increase cortical glutamate levels. As a result, we evaluated the capacity of lamotrigine, a drug reported to reduce presynaptic glutamate release, to reduce ketamine effects in humans.

METHODS: In an ongoing study, healthy subjects (n = 14) completed 4 test days involving the administration of lamotrigine 300 mg, p.o. or placebo 2 hours prior to ketamine (0.23 mg/kg, i.v. bolus and 0.5 mg/kg/hr i.v. infusion) or placebo in a randomized order under double blind conditions. Cognitive, behavioral and neuroendocrine assessments were performed.

RESULTS: Preliminary analysis suggests that lamotrigine blunted the amnestic effects of ketamine, as assessed by the Hopkins Verbal Learning Test (HVLT) (p < 0.05), the perceptual effects of ketamine as assessed by the Clinician-Administered Dissociative States Scale (CADSS) (p < 0.0001), psychiatric symptoms as assessed by the total Brief Psychiatric Rating Scale (BPRS) (p < 0.03) and negative symptoms as assessed by the key negative symptoms sub-scale of the BPRS (p < 0.05). There was a trend for a decrease in the positive symptoms sub-scale of the BPRS (p < 0.07). Ketamine induced a significant mood elevation measured by the Young Mania Rating Scale (YMRS) (p < 0.001). Lamotrigine did not decrease ketamine induced mood elevation. In 8/14 subjects lamotrigine enhanced the mood elevating effects of ketamine.

SIGNIFICANCE: These preliminary data provide indirect support for the hypothesis that the psychotomimetic effects of ketamine are mediated by enhancement of glutamate release. Lamotrigine may be useful in the treatment of schizophrenia, depression, and dissociative disorders.


IS IBOGAINE LIKE MK-801? EVALUATION OF IN VIVO NEUROBIOLOGICAL EVIDENCE (GI-B)
Baumann, M.H.; Rothman, R.B.; and Ali, S.F.

Ibogaine (IBO) is a naturally-occurring indole compound that is being evaluated as a potential medication for substance use disorders. Although the precise mechanism of IBO action is unclear, recent in vitro data show this drug displays properties similar to the non-competitive NMDA antagonist MK-801. The purpose of the present work was to compare in vivo neurobiological effects of IBO and MK-801 in rats. Groups of male rats (N=6-8/group) were decapitated 30 and 60 min after receiving ip IBO (10 & 100 mg/kg), MK-801 (0.1 & 1.0 mg/kg) or vehicle. Trunk blood was collected for the analysis of plasma prolactin and corticosterone; brains were harvested and dissected for determination of DA, 5-HT and their metabolites. Both IBO and MK-801 increased corticosterone secretion, but only IBO elevated plasma prolactin. IBO produced dramatic reductions in tissue DA levels with a concurrent increases in the metabolites, DOPAC and HVA. This profile of IBO-induced changes in DA transmission was observed in the striatum, olfactory tubercle, and hypothalamus. The effects of MK-801 on DA metabolism did not mimic IBO, as MK-801 tended to increase DA and its metabolites. Neither drug appreciably affected 5-HT systems. Our results suggest that the effects of IBO on neuroendocrine function and DA transmission are not due to MK-801-like properties of IBO. Thus, the in vivo mechanism of IBO action can not be explained simply on the basis of antagonism at NMDA receptors.


NMDA RECEPTOR FUNCTION IN MAJOR DEPRESSION (GII-B)
Cappiello, A.; Berman, R.M.; Anand, A.; Charney, D.S.; and Krystal, J.H.

A large body of preclinical research supports the hypothesis that the glutamate system is involved in the mechanism of action of antidepressants, potentially playing a role in the final common pathway. The following study provides preliminary insights on the role of NMDA function in mediating symptoms of major depression.

METHODS: Seven subjects (3 female, 4 males), aged 31 to 55, who met DSM-IV criteria for major depression and 18 healthy subjects, aged 18 to 44 underwent two testing conditions in a balanced, double-blinded manner: following an overnight fast, subjects received intravenous administration of ketamine (0.5 mg/kg) or saline administered over 40 minutes. Clinical ratings of depression, psychosis, dissociative state, and cognitive functioning were assessed serially.

RESULTS: Compared to control subjects, depressed subjects experienced significant ketamine-induced changes that consisted of: (1) greater "high" scores on the visual analog scale; (2) decreased verbal fluency performance; and (3) increased positive and negative symptoms on the BPRS. Furthermore, depressed subjects reported marked improvement in depressive symptoms 72 hours after active versus sham infusion (i.e., 25-item Hamilton Depression Rating Scale scores decreased 13 points vs. increased 2 points, respectively [f=3,74, df=5,60, p=0.01]).

CONCLUSION: Further work is needed to clarify time course of these findings. Preliminary results are consonant with an NMDA model of depression and may suggest a role for NMDA receptor-modulating drugs in the treatment of depression.


NMDA-INDUCED DOPAMINE RESPONSE IS BLUNTED FOLLOWING REPEATED KAPPA-OPIOID RECEPTOR AGONIST TREATMENT (GI-B)
Britton, W.B.; Thompson, A.C.; and Shippenberg, T.S.

Repeated exposure to cocaine results in an enhancement of its reinforcing and psychomotor stimulant effects, a phenomenon referred to as sensitization. The involvement of glutamatergic neurons in the initiation and long-term expression of sensitization has been documented. We have previously shown that kappa-opioid receptor agonists (k-agonists) block the development of cocaine-induced sensitization. This treatment also prevents cocaine-induced changes in dopamine neurochemistry. The role of glutamate in mediating these effects of k-agonists are unknown. Accordingly, this study sought to examine the influence of k-agonists on NMDA-evoked dopamine release in the striatum. Sprague-Dawley rats were implanted with permanent indwelling guide cannula aim at the dorsal striatum (n=25). Following recovery, rats were treated with the selective k -agonist U-69593 (0.32 mg/kg/day x 5 days, s.c.) or Vehicle (20 percent propylene glycol, 1ml/kg/day x 5 days, s.c.). Microdialysis was conducted three days after the last treatment. Dopamine levels in the dorsal striatum were evaluated following intrastriatal perfusion of increasing concentrations of NMDA (0M, 100M, 1mM, 10mM, 30mM). The results showed that NMDA perfusion increased striatal dopamine levels in a dose-related manner. Prior administration of U69593 did not alter basal DA levels within this brain region. However, U-69593 treatment significantly inhibited the stimulatory effects of NMDA on striatal DA overflow. These data demonstrate that repeated k -agonist treatment produces long-term adaptation in NMDA-receptor mediated systems. It is hypothesized that these alterations may be one mechanism by which kappa-opioid receptor agonists inhibit the development and long-term expression of behavioral sensitization to cocaine.


CHRONIC LITHIUM ROBUSTLY PROTECTS CULTURED CNS NEURONS AGAINST NEUROTOXICITY INDUCED BY EXCITOTOXINS (GII-B)
Nonaka, S.; Hough, C.; and Chuang, D-M.

Lithium is best known for its therapeutic efficacy in the treatment of manic depressive illness. The molecular mechanisms underlying its clinical efficacy remain unclear. To explore the novel actions of lithium, we studied its neuroprotective effects on primary cultures of CNS neurons.

We found that long-term exposure to lithium chloride dramatically protects cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity which involves apoptosis mediated by N-methyl-D-aspartate (NMDA) receptors. This neuroprotection of lithium is long-lasting, occurs at therapeutically relevant concentrations with an EC50 of approximately 1.3 mM, and requires treatment for 6-7 days for complete protection to occur. In contrast, a 24-hour treatment with lithium was ineffective. The protection in cerebellar neurons is specific for glutamate-induced excitotoxicity and can be attributed to inhibition of NMDA receptor-mediated calcium influx as measured by 45Ca2+ uptake studies and Fura-2 fluorescence microphotometry. The long-term effects of lithium are not due to down-regulation of NMDA receptor subunit proteins and are unlikely related to its known ability to block inositol monophosphatase activity. Our results suggest that modulation of glutamate receptor hyperactivity represents at least part of the molecular mechanisms by which lithium alters brain function and exerts its clinical efficacy in the treatment for manic depressive illness. These novel actions of lithium also suggest that abnormality of glutamatergic neurotransmission as a pathogenic mechanism underlying bipolar illness warrants future investigation.


TWO CONFIGURATIONS OF ENDOTHELIAL GLUCOSE TRANSPORTER EXPRESSION: A COMMON MECHANISM IN HUMAN BRAIN INSULT (GII-A)
Cornford, E.M.

Dynamic FDG-PET scans of patients with complex partial seizures demonstrated that the zone of hypometabolism (which includes and extends beyond the anatomical lesion) was also a region of significantly reduced glucose influx. Confirmation of this finding was sought with immunogold electron microscopic quantification of the blood-brain barrier (BBB) Glut1 glucose transporter in tissue from seizure resections resected from patients undergoing surgery for treatment of seizures. Samples were prepared from two different regions of each resection: 1) the most actively spiking epileptogenic site, and 2) the least actively spiking region, as indicated by intraoperative EEG monitoring. Two configurations of endothelial cell Glut1 were observed. About one-half of the capillary profiles examined displayed abundant Glut1 immunoreactivity on both luminal and abluminal endothelial membranes. In the remainder of the profiles, reduced Glut1 labeling was seen; but adjacent erythrocyte membranes remained highly Glut1-immunoreactive, suggesting that reduced endothelial Glut1 reactivity was not attributable to method artifacts. Altered glucose transporter activity in the BBB was characterized by a bimodal Glut1 distribution in which the smaller (Type B) endothelial cells displayed low Glut1 immunoreactivity, while adjacent (and even contiguous) larger (Type A) endothelial cells showed 5-10-fold greater expression of membrane Glut1 transporter protein. Immunogold studies using antisera to human glial fibrillary acidic protein (GFAP) and human serum albumin (HSA) demonstrated increased quantities of these two epitopes in the extravascular regions in which more EEG spiking activity had been demonstrated. These observations indicate that capillary integrity was more compromised, and gliosis was quantitatively increased, in the more actively spiking region of the resection. Altered BBB Glut1 expression has also been observed in brain injury, CNS tumors, and Alzheimer's disease; but not in 8 different normal primate brains examined. In conclusion, common response pathways in CNS disease apparently modify brain glucose acquisition, and emphasize the dynamic contributions of BBB capillaries to human brain homeostasis. (Supported by NS-25554.)


PRELIMINARY EVALUATION OF THE TOLERABILITY/SAFETY OF HIGH DOSE DEXTROMETHORPHAN IN METHADONE-MAINTAINED SUBJECTS (GI-B)
Cornish, J.W.; Herman, B.H.; Ehrman, R.N.; Childress, A.R.; Bead, V.; Hackett, C.; Caruso, F.S.; Walsh, R.; Martz, K.; Bloch, D.; Oehlert, J.; Hill, J.; O'Brien, C.P.; Bridge, P.; and Vocci, F.

Results of preclinical research suggest that glutamatergic systems play an important role in opioid dependence (cf. Herman & O'Brien Semin Neurosci 9:158-172, 1997). NMDA receptor antagonists (e.g., dextromethorphan (DM)) inhibit and reverse the analgesic effects of morphine in rodents (cf. Inturrisi Sem Neurosci 9:110-119, 1997). Co-administration of glutamatergic antagonists with opioid medications (methadone (M)) may facilitate the dose reduction of these agents and decrease relapse to abused opiates. The present inpatient study evaluated the safety/tolerability of co-administration of DM + M as a precursor to a possible efficacy trial. Fifteen subjects were randomized in a double-blind (DB) fashion to either DM (N = 10) or Placebo (P) (N = 5). In this 16-day (d) study, DM doses (qid) included: 120-, 240-, and 480-mg/d. Daily M doses were between 50-70 mg/d and were constant. Data are presented for the completers (N = 8 for DM; N = 5 for P). Initial analyses focused on the 480 mg/d DM comparisons and included planned comparisons with P counterparts at 1h following medication for: pulse, systolic blood pressure, respiration rate, temperature and affective measures (ARCI subscales) and at 2h for: visual analog scale (VAS) (opiates, nicotine). During the P run-in phase, both groups had similar cardiovascular, pupil size and affective profiles, however both opiate and nicotine VAS showed a two-fold difference. During the DB medication phase, no significant differences between groups were detected on any variable, including euphoria, dysphoria or VAS (opiate, nicotine). The VAS opiate and nicotine effects are of interest. These results suggest that the addition of large doses of DM (up to 480 mg/d) to a moderate dose of M failed to have significant AEs, indicating the apparent tolerability of DM + M. Further studies evaluating DM + M for the treatment of opiate addiction are projected. (Supported by an Interagency Transfer Agreement Between MDD, NIDA and the Philadelphia VA and a CRADA between MDD, NIDA and ALGOS.)


NMDA RECEPTOR INVOLVEMENT IN ANOXIA-INDUCED DISRUPTION OF IONIC HOMEOSTASIS IN GREY MATTER IN VITRO? (GII-A)
Croning, M.D.R. and Haddad, G.G.

Neuroprotective effects of NMDA receptor antagonists have been demonstrated in animal models of stroke, particularly of the focal ischemia type. However, in brain slices maintained in vitro, subjected to severe hypoxia or anoxia, little, if any, significant reduction is seen in neuronal depolarization or disruption of ionic homeostasis when they are treated with NMDA receptor antagonists.

Therefore we hypothesised that in these slice models, the extracellular acidosis that arises as a result of stimulation of glycolysis during oxygen deprivation may be sufficient to prevent NMDA receptor activation, despite the occurrence of glutamate release and cellular depolarization. In order to test this hypothesis, we subjected rat hippocampal and cortical slices to anoxia with or without concurrent glucose deprivation, and used ion-selective microelectrodes to record alterations in extracellular ion concentrations, monitor the onset of anoxic depolarization, and determined the effect of an NMDA receptor antagonist on these events. We found that reducing glucose availability greatly attenuated the anoxia-induced decrease in extracellular pH both in the CA1 stratum pyramidale of the hippocampus and layers II/III of parietal cortex slices. Under the low glucose, high pH conditions, (Å)-AP-5 (100 M) significantly delayed the onset of anoxic depolarization in both regions. However, it had little effect on the time of onset as a result of anoxia alone. The magnitude of the changes in extracellular [K+] and [Na+] as a result of anoxia Å glucose deprivation were not significantly reduced by (Å)- AP-5, suggesting routes other than the NMDA receptor-ionophore complex, may exist for the fluxes underlying these changes.


KETAMINE EXACERBATES SYMPTOMS IN SCHIZOPHRENIC SUBJECTS: EVIDENCE FOR ALTERED NMDA RECEPTOR FUNCTION? (GII-B)
D'Souza, D.C.; Abi-Saab, W.; Gil, R.; Karper, L.; Charney, D.; and Krystal, J.H.

BACKGROUND: A leading hypothesis suggests that abnormalities in NMDA receptor function contribute to the neurobiology of schizophrenia. It is difficult to study NMDA receptor dysfunction in isolation. The ketamine model provides a direct, clear, interpretable and replicable method to study the consequences of selective deficits in NMDA receptor function and its contributions to the neurobiology of schizophrenia. Ketamine produces schizophrenia-like symptoms in health subjects by noncompetitive NMDA receptor antagonism.

PURPOSE: Do schizophrenics have altered glutamatergic function as evidenced by altered ketamine sensitivity?

METHODS: Neuroleptic-treated schizophrenic subjects completed 2 test days during which they received ketamine (a bolus of 0.65 mg/kg/min for 1 minute followed constant infusion of 0.5 mg/kg/min for 1 hr.) or placebo in a double-blind, randomized and counterbalanced design. Measures of psychosis (Brief Psychiatric rating Scale: BPRS), perceptual alterations, frontal and temporal cortical, and motor function were administered.

RESULTS: Ketamine increased 4-key positive symptom subscale scores (p = 0.07), 3-key negative symptom subscale scores (p = 0.06) and total scores (p = 0.004) of the BPRS. Ketamine also increased clinician-rated (p = 0.0001) and subject-rated (p = 0.0006) perceptual alteration scores. These results were compared to ketamine effects in healthy subjects who were pretreated with haloperidol 5 mg. There were differences in both the magnitude and quality of ketamine effects in neuroleptic-treated schizophrenics and neuroleptic-treated controls. For example, ketamine produced greater changes in scores of perceptual alterations, negative symptoms and positive symptoms in schizophrenics versus controls.

CONCLUSIONS: These data suggest altered ketamine sensitivity in schizophrenia and lend support to the hypothesis that altered glutamatergic function contributes to the neurobiology of schizophrenia. (Supported by the Department of Veterans Affairs.)


TOXICITY OF MUTANT SOD-1 IN MOTOR NEURONS IS MEDIATED VIA NON-NMDA IONOTROPIC RECEPTORS AND PREVENTED BY CALBINDIN D28k (GII-A)
Figlewicz, D.A.; Roy, J.; Minotti, S.; Dong, L.; and Durham, H.D.

Mutations in Cu/Zn-superoxide dismutase (SOD-1) are responsible for a familial form of amyotrophic lateral sclerosis (FALS). We have established a primary culture model which reproduces many features of this disease (J. Neuropathol. Exp. Neurol. 56: 523-530, 1997). Human SOD-1 cDNAs bearing mutations found in FALS patients (mSOD) were subcloned into pCEP4. mSOD constructs or pCEP4 "empty vector" were microinjected into nuclei of motor neurons in dissociated cultures of murine spinal cord, along with the marker dextran-FITC. A gradual loss of motor neurons injected with mSOD constructs occurred over 12 days [G93A (53 percent), G41A (54 percent), N139K (86 percent)]. Blockade of AMPA/kainate receptors by CNQX prevented death of motor neurons expressing mSOD. Since mutant protein was expressed only in motor neurons in this model, dysfunction of astrocytes or of neurons providing synaptic input could not have contributed to motor neuron death. Thus, normally non-toxic levels of glutamate receptor activation were sufficient to potentiate the toxicity of mSOD in motor neurons, particularly via non-NMDA ionotropic receptors.

The presence of Ca++-permeable AMPA receptors has been implicated in the preferential susceptibility of motor neurons in ALS, exacerbated by a deficiency of cytosolic Ca++ - binding proteins such as calbindin. In cultured motor neurons, dramatic neuroprotection was obtained by coinjecting calbindin-D28k and mSOD expression vectors: viability was indistinguishable from control cultures. Partial protection was also obtained by blockade of L-type voltage-gated calcium channels with nifedipine. Our results demonstrate the Ca++-dependence of mSOD-1 toxicity and implicate entry of Ca++ through both glutamate receptor channels and voltage-gated calcium channels in potentiating the toxicity of these SOD mutants in motor neurons.


POTENTIAL INVOLVEMENT FOR THE PLASMA MEMBRANE CALCIUM ATPASES IN GLUTAMATE INDUCED CELL DEATH (GI-A)
Garcia, M.L.; Strehler, E.E.; Isackson P.J.; and Windebank, A.J.

Glutamate toxicity may be the final common pathway of several neuronal insults and neurodegenerative diseases. Delayed cell death, induced by excess glutamate, is mediated by calcium. As an initial assessment of the possible involvement of the plasma membrane calcium ATPases (PMCAs) in glutamate induced neurodegeneration, we have determined the effect of seizures upon PMCA mRNA expression. Kainic acid altered the expression within the hippocampal subfields of all PMCA mRNA isoforms, as determined by in situ hybridization. PMCA 1, 2 and 3 mRNAs exhibited hybridization below control levels 12 to 48 hours post-injection within CA1 and CA3 of the hippocampus. In the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 hours post-injection and exceeded control levels at 48 and 96 hours post-injection. The physiological significance of altering the expression of PMCA isoform 4 is now being investigated in a cell culture model. PC12 cells were transfected with an antisense (pCIneo-antiPMCA4) construct to decrease endogenous PMCA4 expression. In addition, PC12 cells were transfected with a full length sense (CMV-hPMCA4b-neo) construct to overexpress hPMCA4b. Clonal cell lines expressing these constructs have been established. Viability studies will determine the effects of altering PMCA expression on cell survival.


CHANGES IN AMPA RECEPTOR SUBUNIT EXPRESSION IN VENTRAL MOTOR NEURONS AFTER SPINAL CORD INJURY (GII-A)
Grossman, S.D.; Wolfe, B.B.; Yasuda, R.P.; and Wrathall, J.R.

Motor neurons are particularly susceptible to AMPA/KA mediated excitotoxicity. They undergo chronic neurodegeneration in amyotrophic lateral sclerosis and following spinal cord injury (SCI). Further, we found the AMPA/KA antagonist NBQX to be highly neuroprotective in a standardized model of SCI. We have now investigated changes in AMPA receptor subunits in ventral motor neurons following contusive SCI (10g x 2.5cm) at T8. Two groups of rats (n = 3 per group) were analyzed to determine changes in mRNA levels: Acute SCI at 24 hours postinjury (p.i.) and acute laminectomy controls. In situ hybridization was done using four [35S] end labeled antisense oligodeoxynucleotide probes specifically directed against GluR1, GluR2, GluR3, or GluR4. We analyzed grain counts from surviving motor neurons 4mm away from the epicenter of injury, where approximately 60 percent of ventral motor neurons are spared after SCI. We found a significant decrease in GluR2 mRNA (53 percent) at 24 hours p.i. compared to control levels. The decrease in GluR2 is specific since neither GluR3 nor GluR1 mRNA was significantly changed by 24 hours. These results are consistent with changes seen in protein levels, as determined by Western blot of thoracic tissue homogenates at 24 hours p.i., using antibodies against GluR1 and GluR2/3. Since the relative abundance of GluR2 is positively correlated to the calcium impermeability of the channel, acute downregulation could foster excitotoxicity. Alternatively, calcium influx through AMPA receptors may promote plasticity through synaptic strengthening, as is the case in long term potentiation. (Supported by NIH NS28130.)


COMPARISON OF SUBUNIT-SPECIFIC NMDA RECEPTOR CHANNEL BLOCK BY ADCI AND KETAMINE (GI-A)
Harty, P. and Rogawski, M.A.

ADCI ([(+/-)-5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine]), and ketamine (2-O-chlorophenyl-2-methylamino cyclohexanone), reduce NMDA-evoked currents by a channel blocking mechanism. Yet these two compounds exhibit very different behavioral and clinical profiles. Ketamine is a dissociative anesthetic similar to dizocilpine and phencyclidine, producing psychotomimetic effects at high doses. In contrast, ADCI is considered primarily an anti-convulsant, and exhibits little or no psychotomimetic effects. We hypothesized that these marked differences might be related to subunit-specific blocking properties of the two compounds. To test this idea, we used recombinant DNA and mammalian (HEK) cells to express NMDA receptors composed of subunits NR1a/NR2A, NR1a/NR2B or NR1a/NR2C. Using a rapid perfusion system, NMDA receptors of known subunit composition were exposed to NMDA with and without different concentrations of blockers. These data were used to construct and compare the sub unit-specific dose-response properties of the two compounds. In addition, the onsets and offsets of block produced by the drugs at the different heteromeric receptors were fit with exponential functions, and the time constants were incorporated into simple kinetic models of ligand binding.


DEAFFERENTATION INDUCED CELL DEATH IN THE SPINAL DORSAL HORN IS POTENTIATED BY NMDA RECEPTOR STIMULATION BUT NOT BY GABAA OR GLYCINE RECEPTOR INHIBITION (GII-B)
Jasmin, L. and Janni, G.

The neural pathways responsible for nociceptive transmission are not hard wired, but undergo structural and functional changes in response to neural and trophic signals. Events taking place in the periphery can trigger changes that can then modify sensory processing temporarily or permanently in the CNS. Deafferentation injury in particular leads to chronic intractable pain. We present evidence of cell death in nociceptive areas of the spinal cord after peripheral nerve section in adult rats, a phenomenon that could be a determinant in the irreversibility of symptoms and altered responses to therapy. Sciatic nerve section was associated with apoptotic and necrotic neuronal death in the lumbar superficial dorsal horn 24 hours after injury. NMDA (6.9 nmol) administered intrathecally at the time of sciatic n. section was followed by a 300 percent increase in the number of apoptotic cells. NMDA in sham operated rats, however, did not by itself cause any cell death.

Intrathecal administration of the GABAA antagonist bicuculline (40 to 80 nmol) or the glycine receptor antagonist strychnine (80 nmol) had no effect at doses that induce strong nociceptive behaviors. These results indicate that spinal nociceptive neurons acutely stimulated by peripheral nerve injury are more susceptible to NMDA excitotoxicity. It remains to be determined if the commitment to cell death occurs at the time of nerve injury as a result of massive depolarization, or if it occurs later as a result of either stimulus and/or deafferentation induced transcriptional and biochemical events, a phenomenon potentiated by NMDA receptor stimulation.


ACTIVATION OF HIPPOCAMPAL GLUTAMATE RECEPTORS IN A MOUSE MODEL OF AIDS DEMENTIA COMPLEX (GII-A)
Keele, N.B.; Usherwood, P.N.R.; Espey, M.G.; Basile, A.S.; and Rogawski, M.A.

Whole-cell voltage clamp of cultured rat hippocampal neurons in vitro was used to study the effects of antibodies isolated from mice inoculated with the leukemia retrovirus LP-BM5 in vivo, a mouse model of AIDS (MAIDS). MAIDS mice share immunological and neurological effects observed in humans infected with HIV-1, including the neuropathology and cognitive impairments which manifest as AIDS dementia complex (ADC). Over-activation of glutamate receptors is implicated in ADC. This project tests the hypothesis that the MAIDS immune response yields antibodies that activate glutamate receptors in the brain, and characterize the mechanism of action.

The following observations were made:

  1. MAIDS serum IgG and MAIDS brain IgG evoke inward current in hippocampal neurons, but not serum from control mice.

  2. The MAIDS IgG-activated current (Imaids) is inhibited by CNQX, but more potently by the AMPA receptor-selective non-competitive antagonist GYKI 52466.

  3. The reversal potential of Imaids is similar to that of kainate-activated current.

  4. Imaids is sensitive to the AMPA receptor desensitization inhibitor, cyclothiazide (CTZ).

  5. The concentration-response relationship of Imaids is an inverted-U shape, suggesting a CTZ-insensitive component of desensitization.

  6. Imaids is not observed until hippocampal neurons are maintained 9 days in vitro (div) and is apparently maximal at 14 div, suggesting developmental regulation.

  7. Membrane currents evoked by AMPA and MAIDS interact cooperatively.

  8. These data are consistent with retrovirus-induced antibodies that activate AMPA receptors in the brain. The antibody GluR activation, together with potential cooperativity with endogenous ligands, may participate in the excessive glutamatergic tone underlying the neurological deficits of ADC.


METABOTROPIC GLUTAMATE RECEPTORS, MIDBRAIN DOPAMINE AND THE GENERATION OF LOCOMOTOR ACTIVITY (GI-B)
Kim, J. and Vezina, P.

Interactions between excitatory amino acid (EAA) and dopamine (DA) pathways in the basal ganglia have been known for some time to contribute importantly to the generation of motor behaviors. In particular, the role played by ionotropic glutamate receptors (iGluRs) in such interactions and in the production of locomotion has received considerable attention particularly in brain areas such as the ventral tegmental area (VTA) where EAA afferants are known to modulate the activity of DA neurons and the nucleus accumbens (NAcc) where descending EAA projections and ascending DA mesencephalic projections come in close apposition to each other and co-innervate intrinsic neurons projecting to motor output regions. Recently, the growing importance of the metabotropic glutamate receptor (mGluR) in the generation of motor behaviors and various forms of plasticity has begun to emerge. The known coupling of the mGluR to second messenger systems and its demonstrated role in the long-term modulation of synaptic transmission make it a logical candidate not only for the generation of locomotion involving EAA-DA interactions but also for the induction and expression of locomotor plasticity involving these neurotransmitters. Evidence is presented supporting a role for mGluRs in the generation of DA-dependent locomotion as well as in one form of locomotor plasticity: the sensitization of locomotor activity by psychomotor stimulant drugs.


NORMAL SENSITIVITY TO SPERMINE OF NMDA-EVOKED RESPONSES FROM TRISOMY 16 MOUSE EMBRYONIC CULTURED HIPPOCAMPAL NEURONS (GII-A)
Klein, R.C.; Siarey, R.J.; Rapoport, S.I.; Castellino, F.J.; and Galdzicki, Z.

It has been proposed that polyamines (e.g. spermidine, spermine) play an important role in brain development, function, and in certain neurodegenerative conditions (8). Down syndrome (DS) is the most common, human autosomal abnormality characterized by mental retardation, hypotonia, and early onset of Alzheimer's disease (AD). McCoy et al. (1982) (3) reported a decreased content of polyamine in DS lymphocytes stimulated by Concanavalin A. In addition, lower levels of spermidine and spermine were found in frontal cortex of DS and AD patients (5).

The trisomy 16 (Ts16) mouse is considered a genetic model of DS, as there is a substantial homology between the long arm of human chromosome 21 and the distal portion of mouse chromosome 16. No AD-type phenotypic changes were found in Ts16 grafts (6), however functional changes, similar to AD abnormalities, have been reported (2, 4). In addition, changes in densities of voltage-dependent sodium and calcium currents in Ts16 hippocampal cultures have been reported (1, 7).

The NMDA receptor constitutes a second major route of calcium entry into hippocampal neurons, therefore we investigated NMDA-evoked currents and the sensitivity to spermine. In both Ts16 and diploid neurons, NMDA
(6-150 M) evoked similar inward currents. All principle properties, such as the reversal potential, the minimum current, and the dose response of NMDA-evoked currents showed no significant differences between Ts16 and diploid neurons. The current evoked by NMDA was potentiated in a dose dependent manner for all doses of spermine (< 1 mM) and this potentiation was not voltage-dependent. The dose-responses of spermine were similar for both Ts16 and diploid neurons. These results further support our previous data that NMDA-evoked currents are not altered in Ts16 neurons.

References

1. Galdzicki, Z.; Coan, E.J.; Rapoport, S.I.; and Stoll, J. Mol Brain Res, in press, 1998.

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ALTERED RESPONSES TO THE NMDA ANTAGONIST, KETAMINE, IN RECENTLY DETOXIFIED EARLY ONSET ALCOHOLICS (GI-B)
Krystal, J.H.; Petrakis, I.L.; D'Souza, D.C.; Trevisan, L.; Karper, L.; Cooney, N.; and Charney, D.S.

At doses commonly associated with intoxication, antagonism of NMDA glutamate receptors is among the most prominent actions of ethanol. The purpose of this study is to begin to study the clinical implications of the NMDA antagonist actions of ethanol: 1) do alcoholic patients recognize NMDA antagonists as ethanol-like? and 2) are there changes in the sensitivity to ketamine that might be consistent with post-mortem data describing increases in NMDA receptor binding in these patients?

METHODS: Recently detoxified male alcoholic patients (n = 20) and male controls (n = 15) completed 3 test days involving the i.v. administration of placebo, ketamine 0.1 mg/kg, or ketamine 0.5 mg/kg over 40 min. under double-blind conditions.

RESULTS: Ketamine effects were rated by patients as more similar to ethanol than to cocaine or marijuana. The 0.1 mg/kg was rated as similarly in intensity to approximately 1 standard ethanol drink and the 0.5 mg/kg dose was rated as similar in intensity to approximately 9 standard ethanol drinks. The psychotigenic, perceptual, cognitive, and neuroendocrine effects of ketamine were significantly blunted in patients relative to controls. However, the euphoric effects of ketamine were not reduced in patients.

IMPLICATIONS: These data suggest the NMDA antagonist properties of ethanol figure prominently in the behavioral effects of this drug. Reduced ketamine sensitivity in recently detoxified alcoholics may be consistent with cross-tolerance between ethanol and ketamine. Lastly, the reward valence of ketamine appears to be altered in recently detoxified alcoholics: they experience markedly reduced levels of unpleasant ketamine effects (psychotigenic, perceptual, cognitive, anxiogenic) relative to controls, but preserved euphoric effects. This relative enhancement in the overall rewarding properties of ethanol may contribute to heavy drinking, and thus relapse, in recently detoxified patients. (Supported by NIAAA and the Department of Veterans Affairs.)


DISRUPTIVE EFFECTS OF THE NMDA ANTAGONIST, KETAMINE, UPON SENSORIMOTOR GATING IN HUMANS (GII-B)
Krystal, J.H.; Karper, L.; Abi-Saab, D.; Belger, A.; D'Souza, D.C.; Charney, D.S.; and Grillon, C.

Previous clinical research conducted by our group and others suggests that schizophrenia is associated with impairments in sensorimotor gating, i.e., deficits in the "pre-pulse" inhibition (PPI) of the startle response that are associated with impairments in focusing attention, i.e., filtering environmental stimuli. Previous preclinical research has suggested that NMDA receptor deficits contribute to PPI deficits and that these deficits are preferentially reduced by atypical rather than typical neuroleptics. In addition, characterization of ketamine effects on PPI may help to characterize the cognitive effects of drugs, such as ethanol, that block NMDA receptors.

METHODS: Healthy subjects (n = 20) completed 2 test days, involving i.v. infusion of placebo or ketamine (bolus 0.23 mg/kg followed by 0.5 mg/kg/hr). The acoustic startle response was measured (San Diego Instr. Startle-SR) and other assessments were completed. Each startle trial consisted of 6 blocks (collapsed to 3 for analysis) containing 6 stimuli involving all combinations of 3 pre-pulse intensities (no prepulse, 48 dB and 51 dB) and 2 stimulus intensities (100 dB and 113 dB).

RESULTS: Ketamine reduced startle amplitude, but did not significantly effect habituation. The PPI analysis focused on the first of the three blocks of stimuli. Ketamine significantly reduced pre-pulse inhibition of the startle response (drug by prepulse by stimulus intensity interaction by order of test day: F[2,32] = 4.2, p = 0.02). These effects were greatest for the weaker pre-pulse and pulse stimulus intensities where the magnitude of PPI was reduced by approximately one-half (mean placebo: 91 units vs. mean ketamine: 47 units). Cognitive data are currently under analysis.

IMPLICATIONS: These data suggest ketamine depresses the acoustic startle response in humans. In addition, ketamine reduces PPI of the startle response in a fashion that is dependent on several factors. Previous data from our group described increases in distractibility associated with ketamine administration in humans. These data are consistent with the view that deficits in NMDA receptor function that reduce sensory gating may contribute to impairments in the control of attention. (Supported by NARSAD and the Department of Veterans Affairs.)


GLUR5 KAINATE RECEPTOR MEDIATED SYNAPTIC TRANSMISSION IN RAT BASOLATERAL AMYGDALA IN VITRO (GI-A)
Li, H.; Post, R.M.; and Rogawski, M.A.

This study examines a non-NMDA and non-AMPA receptor mediated excitatory postsynaptic response evoked in the basolateral amygdala (BLA) by the stimulation of the external capsule (EC). The 2,3-benzodiazepine AMPA receptor-selective allosteric antagonists GYKI 52466 and GYKI 53655 (50 &M) depressed single shock EC-BLA synaptic responses by 66 Å 3 percent and 83 Å 3 percent respectively but fully blocked synaptic responses evoked by stimulation of basal amygdala (BA). Applications of train stimulation to the EC in the presence of GYKI 52466-containing cocktail and GYKI 53655-containing cocktail evokes a further increase in the amplitude of synaptic response range from 8.4 Å 1.4mV and 7.1 Å 1.1mV respectively. Thus in the presence of GYKI 52466-containing cocktail this train evoked synaptic responses are largely blocked by 20 &M CNQX at 85.3 Å 3.5 percent. Meanwhile, in the presence of GYKI 53655-containing cocktail train evoked synaptic responses are fully blocked by 10 &M LY293558 at 94.5 Å 2.3 percent suggesting this kainate receptor mediated synaptic transmission is mediated predominantly by GluR5 receptor activation in the amygdala.


NMDA RECEPTOR SUBUNITS DISTRIBUTION IN CULTURED CORTICAL NEURON (GI-A)
Li, J. and Vincini, S.

Using antibodies against distinct NMDA receptor (NR) subunits, we report that in developing rat cortical neurons in culture NR1 and NR2B subunits display high levels of expression within the first week while the NR2A subunit is barely detectable at 7 days in vitro (DIV) and gradually increased to mature levels. Immunocytochemical analysis indicated that clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. In some neurons NR subunit clusters failed to develop, and only diffuse microclusters were observed. At DIV18 NR2A and NR2B clusters well colocalize with those of NR1 subunits however the extent of colocalization between NR2A and NR2B subunits was cell specific as the antagonism of NR-mediated synaptic currents by subtype selective drugs. Synapse formation as indicated by synaptophysin staining and electrophysiological recordings, was observed as early as 72 hours after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. We also investigated the distribution of NR subunit clusters in relationship to distinct neuronal compartment such as axon, dendritic shafts and dendritic spines by transient transfection of green flourescent protein (GFP) into cultured cortical neurons. We provide a direct evidence that the NR2B subunit clusterize on both dendritic shafts and spines. At the same time, we also observed that NR2B subunit is poorly colocalize with GluR1 subunit. These data provide a direct evidence of cell and compartment dependent expression and distribution of specific NR subunits.


ACCUMULATION OF GLUTAMATE IN EXPERIMENTAL DIABETIC RETINOPATHY (GI-A)
Lieth, E.; Ratz, M.J.; Tanase, D.; Antonetti, D.A.; Gardner, T.W.; and Barber, A.J.

The early pathophysiology of diabetic retinopathy and the involvement of neural and vascular malfunction are poorly understood. We recently showed that diabetes impairs glial glutamate metabolism in retinas of rats soon after the onset of hyperglycemia. Elevated retinal glutamate can cause excitotoxic damage to ganglion cells of the retina. Here we investigate the possibility that glutamate accumulates in retinas in short-term diabetes. Streptozotocin diabetic rats and age-matched normal rats were housed for 1, 2 or 3 months. Retinas were dissected and homogenized to measure glutamate by luminometry. Also, glial fibrillary acidic protein (GFAP) was measured by ELISA to assess glial reactivity. After 1 month of diabetes no significant increase could be detected either in GFAP immunoreactivity or in retinal glutamate. After 3 months GFAP maximally increases by 5-fold (p < 0.04) and glutamate rises 1.6-fold (p < 0.04). These findings demonstrate for the first time that retinal glutamate rises in short-term diabetes. Elevated retinal glutamate could therefore damage neurons throughout the course of diabetes, eventually leading to vision loss.


GLUTAMATERGIC MODULATION OF kappaB-BINDING TRANSCRIPTION FACTORS: THE NF-kappaB RED HERRING (GI-B)
Mao, X.; Moerman, A.M.; and Barger, S.W.

Studies of kappaB-binding factors in neural tissues have concluded that the transcription factor NF-kappaB is constitutively active in neurons or that its activity is elevated by glutamate. In contrast, we find no evidence of bona fide NF-kappaB DNA-binding activity in neurons under basal or glutamate-stimulated conditions. In highly enriched neuron cultures from hippocampus and neocortex, we detect a constitutive kappaB-binding activity that is distinct from known members of the NF-kappaB family by antigenicity, electrophoretic mobility, physiological regulation, and biochemical behavior. We have tentively named this factor NKBF. The activity of NKBF was rapidly diminished by toxic exposures of glutamate. Activation of both NMDA and non-NMDA ionotropic receptors was required for the full attenuation of NKBF activity. This effect also involved calcium influx and was sensitive to cyclosporin A. Bona fide NF-kappaB activity was induced by glutamate in cocultures containing large numbers of glia but not in glia cultured alone. When kappaB binding activity was assayed separately in the neurons or glia from a coculture, glia accounted for all of the bona fide NF-kappaB activity. These data indicate that the kappaB-binding transcription factor in neurons is negatively regulated by glutamate, in a manner correlated with but preceding neurotoxicity. This conclusion strengthens our previous data indicating that neuronal kappaB-dependent transcription is correlated with neuronal survival in general and excitoprotection in particular. (Sponsored by funds from NINDS (NS35872), the Alzheimer's Association, and the Inglewood Foundation.)


NOVEL NMDA/GLYCINE SITE ANTAGONISTS PREVENT COCAINE-INDUCED TOXICITY AND LOCOMOTOR SENSITIZATION (GI-B)
Matsumoto, R.R.; Brackett, R.L.; Brown, M.; Pouw, B.; Blyden, J.F.; and Kanthasamy, A.G.

No effective treatments exist for cocaine overdose and addiction. Since excitatory amino acid mechanisms may be involved in cocaine toxicity and abuse, novel NMDA/glycine site antagonists (ACEA-1021, ACEA-1031, ACEA-1328, each an "ACEA compound") were tested for their ability to attenuate cocaine-induced behavioral toxicity and locomotor sensitization in male, Swiss Webster mice. Pretreatment with an ACEA compound (0-60 mg/kg, i.p.) prior to a convulsive (60 mg/kg, i.p.) or lethal (125 mg/kg, i.p.) dose of cocaine significantly attenuated cocaine-induced convulsions and lethality. Protection was also produced by other NMDA/glycine site antagonists (7-chlorokynurenic acid, R(+)-HA-966) and structural analogs (DCQX). Structurally-related quinoxalinediones that lack interactions with NMDA/glycine sites (5-nitroquinoxalinedione, NBQX) failed to prevent cocaine-induced toxicity. The NMDA/glycine site agonist D-cycloserine totally prevented the protective effects of the ACEA compounds, confirming an action through NMDA/glycine sites. Significantly, i.p. administration of an ACEA compound after a normally lethal dose of cocaine prevented death in a significant number of animals (50 to 86 percent protection). Deaths were prevented by ACEA-1021 even when it was administered as a post-treatment in mice first exposed subchronically to cocaine for 7 days before receiving a normally fatal cocaine overdose. At doses that themselves did not alter spontaneous locomotion, concomitant administration of the ACEA compounds further prevented the development of locomotor sensitization. Subchronic pre-exposure to an ACEA compound for 5 days also prevented the development of locomotor sensitization, indicating prophylactic potential. Similarly to ACEA-1021 in human clinical trials (see related abstract), the compounds were well-tolerated, and may have the potential to treat cocaine abuse and overdose.


IN VIVO EFFECTS OF BIPERIDEN AND DIAZEPAM ON BRAIN CHOLINERGIC AND AMINERGIC NEUROTRANSMITTERS DURING SOMAN-INDUCED SEIZURES (GI-A)
Morris, J.L.; McDonough, J.H.; Sozio, S.M.; and Shih, T-M.

Guinea pigs, surgically implanted with cortical EEG electrodes and caudate nuclear-microdialysis probes, were administered pyridostigmine 30 minutes before the organophosphorus cholinesterase inhibitor soman [GD] and atropine/2-PAM. Five minutes after seizure onset, an antiseizure ED50 for the GABA agonist diazepam [DZ] or the antimuscarinic biperiden [BP] was given, and animals were separated into seizure not terminated [ON] and seizure terminated [OFF] groups. Soman-increased acetylcholine [ACh] was decreased by DZ and BP after 30 minutes. While ACh levels in DZ ON and OFF animals did not differ, ACh levels in BP OFF animals were decreased from GD alone and BP ON animals. Soman decreased glutamate [Glu] to 60 percent of control for the first 75 minutes after which Glu returned to control. Both DZ and BP decreased Glu in ON and in OFF animals, especially between 2-4 hours of soman exposure, but Glu levels were similar in ON and OFF animals with both drugs. Soman decreased GABA to 70 percent of control with no changes from BP or DZ. These results suggest that BP and DZ may affect caudate nuclear soman-induced seizure activity by other mechanisms in addition to muscarinic blockade and GABA agonism, respectively. (This work was performed while Dr. Morris held a National Research Council-USAMRICD Research Associateship.)


LONG-TERM POTENTIATION IN VIVO INCREASES RAT HIPPOCAMPAL TENASCIN-C EXPRESSION (GI-A)
Nakic, M.; Manahan-Vaughan, D.; Reymann, K.G.; and Schachner, M.

We investigated the expression of the extracellular matrix glycoprotein tenascin-C after induction of long-term potentiation (LTP) by high frequency tetanization (HFT) in the rat dentate gyrus in vivo. Expression of tenascin-C was evaluated at the mRNA and protein levels by in situ hybridization and immunocytochemistry, respectively. Whereas no tenascin-C mRNA was detectable in control animals, an increase in tenascin-C mRNA levels was observed in the granule cell layer of the dentate gyrus 4 hours after HFT. Twenty-four hours after HFT, tenascin-C mRNA returned to control levels. Expression of tenascin-C protein 4 hours after HFT followed that of controls in that tenascin was detectable in the strata oriens and radiatum of CA1, in the molecular layer and within a narrow area at the inner surface of the granule cell layer in the dentate gyrus. However, 24 hours after HFT, additional patches of tenascin-C immunoreactivity were observed in the molecular layer of the dentate gyrus. No increase in tenascin mRNA or protein levels were detected in control animals that received no stimulation, low-frequency stimulation or HFT in the presence of the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5) or the metabotropic glutamate receptor antagonist (R,S)-a-methyl-4-carboxyphenylglycine (MCPG). These observations implicate a role for tenascin-C in N-methyl-D-aspartate and metabotropic glutamate receptor dependent changes accompanying induction and/or maintenance of LTP.

Key words: extracellular matrix, recognition molecule, synaptic plasticity, mGluRs, NMDA, long-term potentiation.


IDENTIFICATION OF NMDA RECEPTOR DOMINANT MUTATIONS AND STUDY OF APOPTOTIC FUNCTION (GI-A)
Rameau, G. and Ziff, E.

Excitotoxic death of neurons occurs following over stimulation of NMDA-type glutamate receptors (NMDAR). I have shown that transient expression of the NMDAR leads to cell death in chinese hamster ovary (CHO) cells. MK-801, a specific NMDAR antagonist, can block this effect. I have quantitated cell killing by measuring the activity of a co-transfected firefly luciferase expression plasmid. To examine the function of the NMDAR, the NR1 subunit was mutated by PCR mutagenesis. Asparagine at amino acid position 616 and serine at position 617 in the receptor pore were substituted for arginine (N616R) and glutamine (S617Q) respectively. These mutations are known from previous work to decrease fluxes of calcium ions. A survival curve obtained by independently expressing the wild type and mutants receptors showed decrease mutant receptor excitotoxicity in comparison to the wild type. Coexpression of mutants with wild type subunits also exerted dominant negative effects and reduced the excitotoxicity of the wild type receptors.

I have also employed this system to examine the role of apoptosis in excitotoxic cell death induced by the NMDAR. I have shown that cells expressing the NMDAR stained positive for annexin V and displayed nuclear DNA-laddering fragmentation that correlate with apoptotic cell death. Similarly, cortical, hyppothalamus and brain stem derived neurons bind Annexin V and produced the same characteristic DNA laddering when exposed to NMDA. In addition, the ICE-protease inhibitors including the baculovirus derived p35 and the pox-virus CramA proteins, block NMDA receptor-dependent cell death in a dose responsive manner, demonstrating that death occurs by an apoptotic mechanism.

I have studied roles of Ca2+-dependent processes and receptor binding proteins in excitotoxicity. Preliminary data have shown that expression of a constitutively activated form of calcineurin increases NMDAR dependent cell death, and this is potentiated further by coexpression of PSD95. These results suggest involvement of calcineurin in the NMDAR-dependent cell death signal mechanism. (Supported by an Aaron Diamond Foundation Postdoctoral Fellowship.)


THE EFFECT OF EAA MODULATORS ON ACUTE NALOXONE PRECIPITATED OPIATE WITHDRAWAL (GI-B)
Rosen, M.I. and Kosten, T.R.

NMDA receptor antagonists have been reported to decrease the development of tolerance and physical dependence in opiate-treated animals; given acutely these agents as well as an AMPA antagonist attenuate opiate withdrawal. However, agents acting at the PCP-site of the NMDA receptor (e.g., MK801, ketamine) show many common features in animal behavioral paradigms, and are likely to have some PCP-like side effects. An alternative approach has been to develop agents that attenuate excitatory amino acid (EAA) activity but do not act directly at the PCP recognition site. Our group has developed an efficient within-subjects methodology to study the effects of EAA modulators on acute opiate withdrawal. Serial naloxone challenge tests in the same opioid dependent subject yield reliable measures of opiate withdrawal. As would be expected for a paradigm sensitive enough to detect an effective anti-withdrawal agent, the naloxone challenge paradigm detected significant attenuation of specific withdrawal signs by clonidine pre-treatment in 7 subjects. Negative controls have not been systematically tested, but gamma-hydroxybutyric acid had minimal effects on precipitated withdrawal in this paradigm. Results will be presented from putative excitatory amino antagonists tested to date in this paradigm including cycloserine (a putative partial agonist at the strychnine-insensitive glycine site of the NMDA receptor), lamotrigine (a marketed anticonvulsant that attenuates glutamate release), and dextromethorphan (a competitive antagonist at the PCP site of the NMDA receptor).


THE ACTIVATION OF METABOTROPIC GLUTAMATE RECEPTORS PROTECTS NERVE CELLS FROM OXIDATIVE STRESS (GI-A)
Sagara, Y. and Schubert, D.

Metabotropic glutamate receptors (mGluRs) have been implicated in a variety of cellular responses to glutamic acid, including the modulation of long term potentiation, glial activation, and excitotoxicity. The work described in this poster extends the role of mGluRs to include protection from oxidative stress induced programmed cell death. The activation of group I mGluRs protects nerve cells via the inositol-1,4,5 triphosphate (IP3) pathway from cell death initiated by oxidative glutamate toxicity. mGluR1 and 5 antagonists potentiate glutamate toxicity, while selective group I agonists are protective. These glutamate analogues regulate IP3 mass accumulation in accordance with their ability to protect cells, and protection appears to take place at the level of glutathione metabolism. Short term exposure of cells to low concentrations of glutamate desensitizes cells to a subsequent challenge from glutamate. Glutamate exposure also upregulates the expression of mGluR5 in HT22 cells and mGluR1 in cortical primary cultures. Group I mGluR agonists also protect cells from death programs initiated by glucose starvation and cystine deprivation. These data show that the activation of group I mGluRs elicits a protective response in neurons which is able to block at least some forms of cell death.


DEVELOPMENTAL PHENCYCLIDINE (PCP) EXPOSURE: SENSORIMOTOR EFFECTS (GII-A)
Scalzo, F.M.

Our laboratory has been investigating the residual motor effects of developmental phencyclidine (PCP) exposure and has identified critical periods of early postnatal development for PCP-induced alterations in the locomotor response to both PCP and MK-801. In an attempt to determine if developmental exposure to PCP results in alterations in sensorimotor function, we measured the reflex modulation of the acoustic startle response in rats following PCP exposure during two periods of development. Rats were treated with saline or 7.5 mg/kg PCP on postnatal days (PNDs) 4-17 or 24-37. Startle responding was measured 9 and 19 days after PCP treatment. Treatment with PCP on PNDs 4-17 resulted in a shorter response latency and decreased prepulse-inhibition (PPI) using, 40, 60 and 80 msec stimulus intervals. Treatment with PCP on PNDs 24-37 did not affect PPI. The results suggest that subchronic neonatal PCP exposure results in alterations in neural systems mediating the startle response and the modulation of the response. (Supported by NIDA Grants DA-06319 & DA-08240.)


CHANGES IN NMDAR1 AND GLUR2 RECEPTOR SUBUNIT POPULATIONS WITH SENSORINEURAL HEARING LOSS IN THE C57BL/6 MOUSE MODEL OF PRESBYCUSIS (GI-A)
Erulkar, J.S. and Schwartz, I.R.

This study sought to identify changes in two ionotropic glutamate receptor subunits in brainstem auditory nuclei in 8wk (normal hearing) and 12m (severely hearing impaired) C57Bl/6 mice, a well documented model of presbycusis (Mikaelian 1979, Willott 1986). We examined the distributions of two subunits: NMDAR1 (an obligate member of functional NMDA receptors) and GluR2 (which confers Ca+ impermeability to the pentameric AMPA receptor). Hearing levels were confirmed with auditory brainstem responses. Animals were perfused with a 4 percent paraformaldehyde, 0.1 percent glutaraldehyde fixative. Cryostat sections of brainstem were incubated with antibodies to the NMDAR1 subunit (PharMingen) and the GluR2 subunit (gift of Dr. Wenthold), processed with the Vector Elite ABC procedure. There were significant differences in the NMDAR1 and GluR2 staining patterns of both neurons and neuropil of layers in the dorsal cochlear nucleus (DCN). The 8wk mice displayed intense NMDAR1 staining of fusiform cells and cells of the deep layer. In contrast, cells in 12m C57s were lightly stained throughout the DCN. The intensity of staining of the molecular layer neuropil also declined. There was little change in the NMDAR1 staining pattern of the octopus cell area (OCA) of the posterior ventral CN. Both groups showed similar staining in the somata and dendritic trees of octopus cells. Two regions receiving primary afferent input (DCN deep layer and OCA) showed different changes in their staining patterns. There was a decrease in the density of GluR2 immunoreactive cells, in the staining intensity of the cell bodies and dendrites of the fusiform layer and in the staining intensity of the neuropil of the molecular layer between 8wk and 12m. Changes in the NMDAR1 and GluR2 staining across the CN did not show a relationship to the underlying tonotopic organization, as might be expected if CN changes were secondary to progressive cochlear degeneration from base to apex. Our data suggest that genetically controlled changes in NMDA and GluR2 receptors may provide an explanation for the observation that deficits in the processing of auditory information in presbycusis is greater than predicted by the peripheral loss (Frisina & Frisina, 1997). (Supported by NIH grant DC00132.)


PRE-SYNAPTIC NMDA RECEPTORS IN GABAERGIC TERMINALS: ULTRASTRUCTURAL EVIDENCE AND SPECIES DIFFERENCE BETWEEN RATS AND MONKEYS (GI-A)
Paquet, M. and Smith, Y.

On the basis of current anatomical and electrophysiological data, NMDA receptors (NMDAR) are commonly seen as post-synaptic receptors. Although some electrophysiological and pharmacological evidence suggest that NMDAR may also have pre-synaptic effects in certain brain regions, immunocytochemical findings revealed that NMDAR subunits immunoreactivity is largely confined to post-synaptic structures. The findings of the present study provide anatomical evidence for the existence of pre-synaptic NMDAR1 subunit immunoreactivity in large subpopulations of axon terminals in the paraventricular (PVH) and arcuate (ARH) nuclei of the hypothalamus, the paraventricular nucleus of the thalamus (PVT) and the bed nucleus of the stria terminalis (BST) in rats. A smaller density of NMDAR1-positive terminals were also found in the basal forebrain, the central nucleus of the amygdala, the rostral hypothalamic region, the ventral tegmental area and the periaqueductal gray.

At the electron microscopic level, NMDAR1-immunoreactive terminals in the PVH, ARH and BST had the ultrastructural features of inhibitory terminals and, indeed, displayed GABA immunoreactivity. It is worth noting that: (1) the NMDAR1 terminal staining was not found in monkeys, and (2) it only showed up with the polyclonal antiserum raised against the C-terminus of the rat NMDAR1 subunit (Petralia et al., J. Neurosci. 14: 667). Two monoclonal antibodies raised against different sites of the NMDAR1 peptide (Siegel et al., PNAS 91: 564; Nash et al., J. Neurochem. 69:485) did not lead to this terminal labelling.

In conclusion, our findings strongly suggest that NMDAR may act as pre-synaptic heteroreceptors in subpopulations of GABAergic terminals in the central nervous system. (Supported by NIH Grant RR-00165 and Dept of Neurology, Emory University.)


MK-801 CAUSES TERMINAL DEGENERATION IN WIDESPREAD AREAS OF RAT BRAIN (GII-A)
Sparenborg, S.; Switzer III, R.C.; Forster, J.; and Filbert, M.G.

Single sc injections of the NMDA antagonist MK-801 (0.1, 0.3, or 0.5 mg/kg) were given to female rats 6, 14, 24, or 96 hours before sacrifice. Neuronal degeneration was evaluated in one brain hemisphere with the cupric-silver method. A single 1-micron-thick methacrylate section from the other hemisphere, containing the retrosplenial cortex, was stained with toluidine blue for visualization of neuronal vacuoles. Rats treated with the two higher doses of MK-801 and sacrificed 6 hours later had vacuoles in the cytoplasm of retrosplenial neurons. The mean number of neurons with vacuoles per section was 0, 0, 10, and 30, respectively, in the control, low, mid and high dose groups. Vacuoles were not found in rats with longer survival times. Although only the higher doses of MK-801 caused vacuolation, all doses caused synaptic terminal degeneration (TD) in many brain areas. At only 6 hours after drug administration, TD appeared in the superficial (I-IV) layers of retrosplenial cortex, in occipital and parietal areas processing vision and audition, and in frontal (motor) association areas. In the hippocampus, TD was mild to severe in the molecular layer of the dentate gyrus and in stratum lacunosum moleculare of CA1. These effects generally increased in severity with longer survival times and were mildly dose-related. Dead neurons were noted in the piriform cortex of some rats given the low dose of MK-801 and sacrificed 14 or 24 hours later. However, silver staining was virtually absent from all brain areas of low-dose rats sacrificed after 96 hours indicating that degenerated terminals and soma had been cleared from the brain. Many dead cell bodies were visible in retrosplenial cortex of mid- and high-dose rats sacrificed after 24 and 96 hours and were occasionally found in piriform and entorhinal cortices. TD represents the permanent loss of inter-neuronal communication through the affected synapses. Widespread TD and the loss of cells in the piriform cortex indicate that MK-801 produced a long-lasting toxic effect at a dose lower than that which produced vacuoles. (Supported by USAMRICD.)


KAINATE ANTAGONISTS AND CHRONIC PAIN (GII-B)
Gu, Z.Q.; Kajander, K.; Maccecchini, M.; and Sturgess, M.A.

Previous efforts at Bearsden Bio (formerly Symphony Pharmaceuticals) had identified (2S, 4R)-4-methylglutamate (SYM 2081) as a novel and potentially promising kainate receptor antagonist. Recent work in our laboratories has developed our understanding of the interaction of this compound with kainate receptors. Data will be presented that show that kainate receptors are a promising therapeutic target for the treatment of chronic pain, and that SYM 2081 is a novel and effective agent for attenuating sensitivity in established animal models of acute and chronic pain.


CELLULAR NEUROANATOMY OF NMDA NR1 SPLICE VARIANTS (GI-A)
Tanaka, M.; Caudle, R.M.; Benoliel, R.; Finegold, A.A.; and Iadarola, M.J.

The primary transcript of the NMDA NR1 subunit undergoes alternative splicing at exons 5,21,22, also called N1 cassette, C1 cassette, C2 cassette respectively, leading to different sets of subunits with different physiological properties. In order to study the distributions and the functions in vivo, we generated polyclonal antibodies affinity purified against exons 5,21,22, and the alternative C-termini when the exon 22 is spliced out. Immunocytochemical staining with these antibodies against the splice variants showed distinct localization. The immunoreactivity of N1 cassette were restricted to the perikarya and the proximal dendritic shaft both in spinal cord and brain. N1 positive cells were located throughout spinal cord and motorneurons were stained strongly. Subunits with the alternative C-termini (C2-) showed a similar subcellular distribution: the cell bodies and proximal dendrites.However , in spinal cord the C2- positive cells clustered around central canal and the motorneurons did not show immunoreactivity. In contrast, C2 containing subunits exhibited a wide spread localization throughout positive cells. This was especially evident in the purkinje cells in cerebellum where immunoreactivity was observed in the perikarya to the ends of the dendritic arbor.Subunits containing the C1 cassette were nearly exclusively expressed in the dendrites of the brain and very low to absent in spinal cord. These data suggest a selective recruitment of NR1 subunits to specific and generalized subcellular distributions in neurons that may be determined by alternative splicing not only of exon 21 but also of exons 5 and 22.


GLUTAMATE CASCADE FROM METABOTROPIC GLUTAMATE RECEPTOR TO IMMEDIATE EARLY GENE EXPRESSION IN RAT STRIATAL NEURONS: IMPLICATION FOR PSYCHOSTIMULANT DEPENDENCE (GI-B)
Mao, L. and Wang, J.Q.

Metabotropic glutamate receptors (mGluR) are coupled to multiple intracellular second messenger systems through G-proteins and expressed by medium spiny projection neurons in the rat striatum. Unlike ionotropic glutamate receptors which mediate rapid synaptic transmission, mGluRs are more important for relatively long-lasting modulation of neuronal metabotropic activities, including gene expression. In chronically-cannulated rats, microinjection of a selective mGluR agonist, ACPD, into the rat striatum elevated immediate early gene c-fos and zif/268 mRNA expression in the injected dorsal striatum as revealed by quantitative in situ hybridization. The elevation of either mRNA was dose-dependent and the induction of c-fos at each dose surveyed was more prominent than that of zif/268. No behavioral alteration was observed after ACPD injection at any dose. Two mRNA induction was rapid and transient, as their increases became evident as early as 30 min, reached a peak at 1 h, and returned to normal levels 3 (c-fos) or 6 (zif/268) h, after ACPD injection. Selective mGluR antagonist, MCPG, was able to attenuate ACPD-stimulated c-fos, but not zif/268, expression. Pretreatment with the ionotropic NMDA receptor antagonist, CPP, had no effect on ACPD-stimulated c-fos expression, but partially attenuated ACPD-stimulated zif/268 expression. D1 dopamine receptor antagonist, SCH23390, did not alter the ability of ACPD to induce the 2-gene expression. The data demonstrate a difference between striatal c-fos and zif/268 gene expression in response to specific mGluR activation. The c-fos elevation was independent of D1 dopaminergic and NMDA glutamatergic transmission whereas zif/268 induction was mediated, at least in part, by NMDA receptors. These results suggest a prominent involvement of mGluRs in cellular gene expression which is thought to be an essential component for neuroplasticity underlying long-lasting action of psychostimulant exposure. (Supported by NIDA DA10355 and a Young Investigator Award from NC Governor's Institute and a research grant (4150486810) from Latham Trust of Crestar Bank.)


METABOTROPIC GLUTAMATE RECEPTORS AS NOVEL TARGETS OF THERAPEUTIC AGENTS FOR SUBSTANCE ADDICTION (GI-B)
Wang, J.Q.

Metabotropic glutamate receptors (mGluR) are densely expressed by striatonigral and striatopallidal projection neurons in the striatum. Activation of mGluRs in this brain region alters local transmitter release and behaviors of experimental animals. Of particular interest is that mGluRs regulate transcription factor and neuropeptide gene expression in the striatal neurons through their connections with multiple intracellular effectors. This prominent involvement of mGluRs in overall cellular activity is significant for development of neuroplasticity underlying long-term adaptive changes in cellular physiology related to a variety of neurological disorders. In our recent studies concerning role(s) of mGluRs in mediation of psychostimulant amphetamine actions, we found that bilateral icv injection of mGluR antagonist, MCPG, markedly attenuated acute amphetamine-stimulated transcription factors, c-fos and zif/268, mRNA expression in the rat striatum, with sparing amphetamine- stimulated behaviors. Parallel with the blockade of the two transcription factors, amphetamine-stimulated mRNA expression of neuropeptides, preprodynorphin, substance P and preproenkephalin, in the striatum was also reduced by MCPG. MCPG itself, however, had little or no effect on constitutive expression of these genes in the striatum and spontaneous behavioral activity. These data indicate a preferential participation of MCPG-sensitive mGluRs in psychostimulant-stimulated gene expression which is believed to be important for neuroplasticity underlying amphetamine dependence. It is notable that this type of glutamate receptor, as the modulatory receptor in nature, has little impact on fast signal transmission in synapse. Thus, mGluR antagonist, as opposed to ionotropic glutamate receptor antagonists, should cause no major general depression or cognitive side effect in chronic therapy. Along with rapid progress in improving brain penetration and subtype specificity, mGluR agents might serve better therapeutic drugs for substance dependence and addiction. (Supported by NIDA/NIH DA10355 and a Young Investigator Award from NC Governor's Institute and a research grant (4150486810) from Latham Trust of Crestar Bank.)


LICOSTINEL: POTENTIAL SOLUTIONS TO SAFETY CONCERNS (GI-A)
Weihmuller, F.

Licostinel (ACEA 1021) is a potent competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor at the glycine site. The strength of safety and efficacy data in animal models has supported the clinical investigation of licostinel for the treatment of ischemic stroke. After obtaining clinical safety data during short (15 min.) infusions of licostinel in 64 stroke patients and 62 healthy volunteers, further investigation of prolonged continuous infusion of licostinel showed formation of crystals in the urine of some subjects. Studies in animal models have shown that the plasma concentration of licostinel at which crystals form in the urine of human subjects was greater (9 times) than the minimum effective plasma concentration (2 ug/ml) required for neuroprotection in stroke models. Furthermore, animal studies have demonstrated that pretreatment with the uricosuric agent probenecid reduces renal excretion and raises plasma levels of licostinel. These findings suggest that a broad margin of safety exists between the minimum efficacious dose and dose-limiting safety concerns. These studies and their implications for future clinical development will be discussed.


GLUTAMATE RECEPTORS REGULATE THE CONVULSANT EFFECTS OF COCAINE (GI-B)
Witkin, J.M. and Gasior, M.

Convulsions associated with cocaine abuse can be life-threatening and resistant to treatment. Status epilepticus following cocaine poisoning is often resistant to standard therapy and can be fatal. In some models of cocaine seizures, even high, incapacitating doses of anticonvulsant drug standards are ineffective against cocaine convulsions. In contrast, functional antagonists of glutamatergic transmission protect against the toxic consequences of cocaine overdose. NMDA receptor antagonists dose-dependently prevent the clonic convulsions induced by cocaine in rodent models. The role of NMDA receptors in the control of cocaine-induced convulsive activity is strengthened by the finding that anticonvulsant activity can be achieved by blockade of both competitive and uncompetitive modulatory sites on the NMDA receptor complex. Thus, competitive antagonists, ion-channel blockers, polyamine antagonists, and functional blockers of the strychnine-insensitive glycine modulatory site of the NMDA receptor complex all prevent cocaine seizures. Although some NMDA blockers produce profound side-effects, significantly reducing therapeutic indices, others (e.g., low-affinity channel blockers, glycine antagonists) demonstrate significant separation in their anticonvulsant and side-effect profiles. The anticonvulsant efficacy of these compounds can be augmented by diazepam suggesting the potential clinical utility of such drug combinations. Interestingly, functional NMDA antagonists may also be of value in the treatment of cocaine dependence.


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