β-Amyloid-Induced Cholinergic Denervation Correlates with Enhanced Nitric Oxide Synthase Activity in Rat Cerebral Cortex: Reversal by NMDA Receptor Blockade

Ample experimental evidence indicates that acute β-amyloid infusion into the nucleus basalis of rats elicits abrupt degeneration of the magnocellular cholinergic neurons projecting to the cerebral cortex. In fact, involvement of a permanent Ca²⁺ overload, partially via N-methyl-

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-aspartate (NMDA) receptors, was proposed as a pivotal mechanism in β-amyloid-induced neurodegeneration. A definite measure of NMDA receptor-mediated processes and subsequent Ca²⁺ entry is the induction of Ca²⁺/calmodulin-activated neuronal nitric oxide synthase (nNOS) in nerve cells. In the present account we therefore assessed activation of nNOS in correlation with cholinergic decline after β-amyloid_(1–42) or β-amyloid_(25–35) infusion into the rat nucleus basalis. The results demonstrate the β-amyloid conformation-dependent enhancement of cortical nitric oxide synthase (NOS) activity. Furthermore, chronic application of the polyamine site NMDA receptor blocker ifenprodil effectively attenuated β-amyloid neurotoxicity. We propose that nNOS activation reflects the degree of β-amyloid-induced excitotoxic injury in a proportional manner. Moreover, Ca²⁺-mediated processes via NMDA receptors, or direct binding of β-amyloid to this receptor may be a critical step in the neurotoxic mechanisms in vivo.     

Selective metabotropic receptor agonists distinguish non-ionotropic glutamate binding sites

Metabotropic glutamate receptors (mGluRs) are thought to mediate diverse processes in brain including synaptic plasticity and excitotoxicity. These receptors are often divided into three groups by their pharmacological profiles. [³H]Glutamate binding in the presence of compounds selective for ionotropic glutamate receptors can be used as a general assay for these receptors; subtypes of this non-ionotropic [³H]glutamate binding differ in both pharmacology and anatomical distribution, and are differentially sensitive to quisqualate. The characteristics of these binding sites are consistent with those of group 1 (high-affinity quisqualate) and group 2 (low-affinity quisqualate) mGluRs. Under our assay conditions, no [³H]glutamate binding to group 3-like (

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-AP4 sensitive) sites could be demonstrated. We have attempted to characterize particular agents which may selectively measure [³H]glutamate binding to mGluR subtypes. We used two isomers of 2-(carboxycyclopropyl)glycine,

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-CCG-I and

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-CCG-II, and the (2S,1′R,2′R,3′R) isomer of 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) as competitors of non-ionotropic [³H]glutamate binding sites. DCG-IV clearly distinguishes two binding sites. Quantitative levels of DCG-IV binding by anatomic region correlate with quisqualate-defined binding subtypes: high-affinity DCG-IV binding correlates with low-affinity quisqualate binding, whereas low-affinity DCG-IV binding correlates with high-affinity quisqualate binding.

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-CCG-II displaces only one type of non-ionotropic [³H]glutamate binding, corresponding to high-affinity quisqualate binding. Therefore DCG-IV and

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-CCG-II at appropriate concentrations appear to distinguish binding to putative group 2 vs. group 1 mGluRs.

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-CCG-I displaces both high- and low-affinity quisqualate binding sites, but unlike the other two compounds, does not clearly distinguish between them.     

Phosphorylation of the Third Intracellular Loop of the Mouse α1b-Adrenergic Receptor by cAMP-dependent Protein Kinase

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases.

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-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse

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-,

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-, and

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-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the

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sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.     

Unique properties of [

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 μM) for 8–24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), both of which are α₇-neuronal receptor antagonists, and dihydro-β-erythroidine (DHβE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α₇-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca²⁺ influx.     

Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α7-neuronal receptors and neuronal CNS receptors

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 μM) for 8–24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), both of which are α₇-neuronal receptor antagonists, and dihydro-β-erythroidine (DHβE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α₇-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca²⁺ influx.     

l-DOPA neurotoxicity is mediated by glutamate release in cultured rat striatal neurons

The exposure of cultured rat striatal neurons to

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-DOPA caused marked cell death. The

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-DOPA cytotoxicity was inhibited by the addition of Mg²⁺ to and by the removal of Ca²⁺ from the culture medium, and also by the application of tetrodotoxin. Moreover, prolonged application of

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-DOPA increased the glutamate content in the culture medium. These results indicate that

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-DOPA produces neurotoxicity by facilitating glutamate release.     

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices

As alterations in intracellular pH (pH_i) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH_i in rat hippocampal slices using the BCECF fluorescent technique. The ‘resting' pH_i in the CA1 pyramidal cell layers was 6.93±0.07 (mean±S.D., n=72 slices) in 25 mM HCO₃⁻/5% CO₂-buffered solution at 37°C. Exposure of hippocampal slices to NMDA in the range of 10–1000 μM produced a biphasic change in pH_i: an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 μM) but not CNQX (40 μM) blocked the NMDA-induced changes in pH_i. In 0 Ca medium (0 mM Ca²⁺ supplemented 1 mM EGTA, referred to as 0 Ca), pH_i acid shift caused by NMDA (20 μM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH_i acid shift by two-thirds, while the NMDA-induced pH_i alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH_i acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH_i. The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca²⁺]_i. The pH_i acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.     

Inhibition of NMDA-induced increase in brain temperature by N-ω-nitro-

Since ATP has been reported to be a potent excitatory transmitter in the mammalian central nervous system (CNS), we studied the neurochemical characters of the binding sites of

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,

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-methylene ATP, an agonist of P_2X receptors, in mouse crude synaptic membranes. ATP and its related compounds inhibited [³H]

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,

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-methylene ATP binding in a concentration-dependent manner. The potency order in the inhibition of the binding was as follows;

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,

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-methylene

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>

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> ATP ≥ ADP >

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,

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-methylene ATP UTP > 2-methylthio ATP. And adenosine did not affect the binding. The order was different from those reported in peripheral tissues. And Sr²⁺, Ca²⁺, Mg²⁺, and Cd²⁺ enhanced the binding. These results suggest that

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,

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-methylene ATP binding sites in CNS have different characters from those in peripheral tissues.     

Characterization of α,β-Methylene ATP Binding Sites in Mouse Crude Synaptic Membranes

Since ATP has been reported to be a potent excitatory transmitter in the mammalian central nervous system (CNS), we studied the neurochemical characters of the binding sites of

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,

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-methylene ATP, an agonist of P_2X receptors, in mouse crude synaptic membranes. ATP and its related compounds inhibited [³H]

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,

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-methylene ATP binding in a concentration-dependent manner. The potency order in the inhibition of the binding was as follows;

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,

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-methylene

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>

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> ATP ≥ ADP >

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,

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-methylene ATP UTP > 2-methylthio ATP. And adenosine did not affect the binding. The order was different from those reported in peripheral tissues. And Sr²⁺, Ca²⁺, Mg²⁺, and Cd²⁺ enhanced the binding. These results suggest that

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,

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-methylene ATP binding sites in CNS have different characters from those in peripheral tissues.     

Monoamine oxidase-dependent metabolism of dopamine in the striatum and substantia nigra of

Glycine is a requisite cofactor for glutamatergic activation of the N-methyl-

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-aspartate (NMDA) receptor. Antagonism of glutamate at the NMDA receptor has been shown to cause substantial changes in regional cerebral metabolic rate for glucose utilization (CMRglu) and blood flow (CBF). This study examined CMRglu and CBF changes caused by antagonism of glycine at the NMDA receptor recognition site. Rats were anesthetized with halothane and vascular access was obtained. The animals were then awakened. One hour later, either vehicle (control) or ACEA 1021 (5 mg/kg followed by 3.5 mg·kg⁻¹·h⁻¹ or 10 mg/kg followed by 7 mg·kg⁻¹·h⁻¹) was infused intravenously. CMRglu and CBF were then determined. Autoradiographic analysis of 25 regions revealed effects of ACEA 1021 on CMRglu in the frontal, sensory, parietal and auditory cortices and the anteroventral and subthalamic nuclei. These changes deviated less than 15% from control. Effects on CBF were also small. The CMRglu and CBF effects of ACEA 1021 are substantially less than those previously observed for either competitive or non-competitive glutamate NMDA antagonists. We conclude that inhibition of the NMDA glycine recognition site has little or no effect on CMRglu or CBF at the doses examined. This is consistent with the absence of psychotomimetic effects observed for this class of drugs.     

Effects of NMDA receptor glycine recognition site antagonism on cerebral metabolic rate for glucose and cerebral blood flow in the conscious rat

Glycine is a requisite cofactor for glutamatergic activation of the N-methyl-

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-aspartate (NMDA) receptor. Antagonism of glutamate at the NMDA receptor has been shown to cause substantial changes in regional cerebral metabolic rate for glucose utilization (CMRglu) and blood flow (CBF). This study examined CMRglu and CBF changes caused by antagonism of glycine at the NMDA receptor recognition site. Rats were anesthetized with halothane and vascular access was obtained. The animals were then awakened. One hour later, either vehicle (control) or ACEA 1021 (5 mg/kg followed by 3.5 mg·kg⁻¹·h⁻¹ or 10 mg/kg followed by 7 mg·kg⁻¹·h⁻¹) was infused intravenously. CMRglu and CBF were then determined. Autoradiographic analysis of 25 regions revealed effects of ACEA 1021 on CMRglu in the frontal, sensory, parietal and auditory cortices and the anteroventral and subthalamic nuclei. These changes deviated less than 15% from control. Effects on CBF were also small. The CMRglu and CBF effects of ACEA 1021 are substantially less than those previously observed for either competitive or non-competitive glutamate NMDA antagonists. We conclude that inhibition of the NMDA glycine recognition site has little or no effect on CMRglu or CBF at the doses examined. This is consistent with the absence of psychotomimetic effects observed for this class of drugs.     

Characterization of [

BIDN (3,3-bis(trifluoromethyl)bicyclo[2,2,1]heptane-2,2-dicarbonitrile) at 10⁻⁵ M blocked GABA-induced inhibitory postsynaptic potentials (IPSPs) recorded from an identified, giant interneurone (G12) of the cockroach (Periplaneta americana). The same concentration of this bicyclic dinitrile also blocked Cl⁻-mediated responses of G12 to GABA applied by pressure microinjection into the terminal abdominal ganglion neuropile containing dendrites of G12. BIDN (10⁻⁵ M) was without effect on a response of G12 to GABA known to be mediated by a GABA_B type receptor. In studies of the cell body of an identified motor neurone, the fast coxal depressor (D_f) in the cockroach metathoracic ganglion, BIDN (10⁻⁵ M) blocked reversibly an extrasynaptic GABA-gated Cl⁻ channel, but not an extrasynaptic

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-glutamate-gated Cl⁻ channel. Glycine-gated Cl⁻ channels observed when rat brain messenger RNA was expressed in Xenopus laevis oocytes were unaffected by BIDN at concentrations up to 10⁻⁴ M, whereas this same concentration of BIDN completely blocked GABA-gated Cl⁻ responses recorded from the same preparations. Unlike picrotoxin, which antagonises a variety of ligand-gated Cl⁻ channels, to date BIDN has been found to block only Cl⁻ channels gated by GABA, both in insect and vertebrate preparations.     

BIDN, a bicyclic dinitrile convulsant, selectively blocks GABA-gated Cl channels

BIDN (3,3-bis(trifluoromethyl)bicyclo[2,2,1]heptane-2,2-dicarbonitrile) at 10⁻⁵ M blocked GABA-induced inhibitory postsynaptic potentials (IPSPs) recorded from an identified, giant interneurone (G12) of the cockroach (Periplaneta americana). The same concentration of this bicyclic dinitrile also blocked Cl⁻-mediated responses of G12 to GABA applied by pressure microinjection into the terminal abdominal ganglion neuropile containing dendrites of G12. BIDN (10⁻⁵ M) was without effect on a response of G12 to GABA known to be mediated by a GABA_B type receptor. In studies of the cell body of an identified motor neurone, the fast coxal depressor (D_f) in the cockroach metathoracic ganglion, BIDN (10⁻⁵ M) blocked reversibly an extrasynaptic GABA-gated Cl⁻ channel, but not an extrasynaptic

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-glutamate-gated Cl⁻ channel. Glycine-gated Cl⁻ channels observed when rat brain messenger RNA was expressed in Xenopus laevis oocytes were unaffected by BIDN at concentrations up to 10⁻⁴ M, whereas this same concentration of BIDN completely blocked GABA-gated Cl⁻ responses recorded from the same preparations. Unlike picrotoxin, which antagonises a variety of ligand-gated Cl⁻ channels, to date BIDN has been found to block only Cl⁻ channels gated by GABA, both in insect and vertebrate preparations.     

Sustained pharmacological inhibition of nitric oxide synthase does not affect the survival of intrastriatal rat fetal mesencephalic transplants

The objective of the present study was to investigate the potential role of the free radical nitric oxide (NO) in the development of fetal rat mesencephalic neurons grafted in a 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease. First, using nitric oxide synthase (NOS)-immunocytochemistry and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, we investigated the presence of the neuronal isoform of NOS (nNOS) in intrastriatal mesencephalic grafts. During the course of the experiment (16 weeks) an increase in the staining intensity and the number of nNOS/NADPH-d positive cells within the grafts was observed, as well as a gradual maturation of dopaminergic neurons. In addition, within both the host striatal and grafted mesencephalic tissue, a NO-dependent accumulation of cyclic guanosine monophosphate (cGMP) was detected, indicating the presence of guanylate cyclase, i.e., the target-enzyme for NO. Secondly, to determine the impact of NO on the survival of grafted dopaminergic neurons, 6-OHDA lesioned rats received mesencephalic grafts and were subsequently treated with the competitive NOS-inhibitor N^ω-nitro-

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-arginine methylester (

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-NAME). After chronic treatment for 4 weeks, tyrosine hydroxylase immunocytochemistry revealed no apparent differences between the survival of grafted dopaminergic neurons in control- or

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-NAME treated animals, respectively. As the maturation of grafted dopaminergic neurons coincides with a gradual increase in the expression of nNOS within the graft and since dopaminergic cell numbers are not changed upon administration of

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-NAME, it is concluded that endogenously produced and potentially toxic NO does not affect the survival of grafted fetal dopaminergic neurons.     

Regional distribution and ionic requirement of Cl/Ca-activated and Cl/Ca-independent glutamate receptors in rat brain

Recent studies have shown that Cl⁻ and Ca²⁺ ions increase [³H]glutamate binding to rat forebrain synaptic plasma membranes by expressing a new class of glutamate receptors. We examined the regional distribution of these two classes of glutamate binding sites and further characterized their ionic requirements. Significant differences in both Cl⁻/Ca²⁺-independent (basal) and Cl⁻/Ca²⁺-activated receptors, as well as the ratios of these two receptor classes were observed among different areas of the CNS. Cl⁻ and Ca²⁺ appeared to act synergistically, with Cl⁻ ion an absolute requirement for Ca²⁺ stimulation, in expressing these additional binding sites. Ca²⁺ alone did not affect glutamate binding.     

Axon sparing lesions of the medial preoptic area block female sexual behavior

The role of the medial preoptic area (mPOA) in regulating female musk shrew sexual behavior was assessed with excitatory neurotoxin, N-methyl-

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-aspartate (NMDA) lesions. Ovariectomized, testosterone-implanted females that received lesions in the mPOA were statistically less likely to show complete sex behavior as compared to controls. These data suggest that the mPOA plays an activational role in testosterone-induced female sexual behavior.     

Distribution of d-amino acid oxidase (DAO) activity in the medulla and thoracic spinal cord of the rat: implications for a role for d-serine in autonomic function

The activity and regional distribution of

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-amino acid oxidase (DAO), an enzyme that inactivates

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-serine, were examined in the medulla and spinal cord of the rat by biochemical and histochemical procedures. DAO activity was noticeably low or absent in the nucleus of the solitary tract, ventrolateral medulla and intramediolateral cell column of the spinal cord. This may be indicative of a neuromodulatory role for endogenous

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-serine (at the NMDA-glycine site) in the central control of blood pressure.     

Anti-epileptogenic and anticonvulsant activity of l-2-amino-4-phosphonobutyrate, a presynaptic glutamate receptor agonist

The protective effect of amygdaloid (focally administered) doses of the presynaptic metabotropic glutamate receptor agonist, l-2-amino-4-phosphonobutyrate (l-AP4) was tested on the development of electrical kindling and in fully kindled animals. l-AP4 inhibited epileptogenesis at 10 nmol in 0.5 μl buffer, by preventing the increase in both seizure score and afterdischarge duration. The effects were reversible after withdrawal of the drug, with all treated animals subsequently progressing to the fully kindled state at the same rate as control animals. The same concentration of the drug was also effective when injected into fully kindled animals. It significantly decreased the mean seizure score by 88% (P<0.005) and increased the mean generalized seizure threshold (GST) by 85% (P<0.005). The increase in GST was accompanied by a significant delay before the onset of generalized seizure and by a 37% reduction in generalized seizure duration. MPPG ((RS)-α-methyl-4-phosphonophenyl glycine) a selective antagonist of l-AP4 at glutamate pre-synaptic receptors inhibited the depressant effect of l-AP4 in a dose-dependent manner. MPPG (10 nmol) inhibited the antiseizure activity of l-AP4, whilst MPPG (40 nmol) reduced both the anti-epileptogenic and antiseizure activities of l-AP4. MPPG (40 nmol) by itself had no effect on generalized seizure activity, and it had no detectable influence on the normal rate of kindled epileptogenesis. During in vitro studies using a microsuperfusion method, l-AP4 inhibited depolarization-induced release of [³H]d-aspartate from rat cortical synaptosomes (IC₅₀ 125.1 μM) and decreased the depolarization-evoked uptake of ⁴⁵Ca²⁺ in a dose-dependent manner. Both actions of l-AP4 were reduced by the selective antagonist MPPG. When applied alone MPPG (200 μM) had no detectable action on veratridine-evoked ⁴⁵Ca²⁺ uptake by the synaptosomes. These results suggest the mechanisms by which presynaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kindling, and indicate that their anticonvulsive activity is due to inhibition of presynaptic glutamate and/or aspartate release following blockade of presynaptic Ca²⁺ entry.     

Effects of acute and repeated administration of N-methyl-d-aspartate (NMDA) into the ventral tegmental area: locomotor activating effects of NMDA and cocaine

Repeated, intermittent administration of psychostimulants produces an enhancement of the subsequent behavioral effects of these drugs. This behavioral sensitization has been implicated in maintenance of and relapse to drug-taking. As a result, there has been great interest in elucidating the mechanisms underlying both the development and expression of sensitization. An accumulation of data from studies of stimulant-induced locomotor activity has implicated excitatory amino acids in the development of behavioral sensitization. In the present study, N-methyl-

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-aspartate (NMDA) (0.6, 1.25 or 2.5 μg) infused bilaterally into the ventral tegmental area (VTA) produced dose-dependent locomotor activation. The locomotor activating effect of NMDA was increased following repeated NMDA administration (two exposures to intra-VTA NMDA), suggesting sensitization. However, repeated intra-VTA NMDA failed to sensitize rats to the locomotor activating effects of systemically administered cocaine (5.0, 10.0 or 20.0 mg/kg). These findings are consistent with the notion that repeated activation of NMDA receptors is sufficient for the development of behavioral sensitization to NMDA. Other neuroadaptations produced by repeated psychostimulant administration are required in order for the development of sensitization to the behavioral effects of those drugs.