Interaction between N-methyl-d-aspartate (NMDA) and serotonin (5-HT) on ethanol tolerance

Earlier work from this laboratory had shown that 5-HT is involved in the development of tolerance to ethanol, and that enhancement of 5-HT levels by l-tryptophan accelerated tolerance development. To explore the possibility that NMDA receptors are involved in the 5-HT effect on tolerance, we examined the effect of a noncompetitive N-methyl-d-aspartate (NMDA) antagonist [(+)MK-801] on the ability of l-tryptophan to enhance tolerance to the effect of ethanol on tilt-plane test performance by the rat. l-Tryptophan treatment resulted in the development of rapid tolerance to a dose of ethanol that failed to produce such tolerance by itself. However, prior administration of (+)MK-801 blocked the l-tryptophan effect on rapid tolerance development, in a dose-dependent manner. These results suggest that NMDA receptors are involved in the 5-HT enhancement of ethanol tolerance.     

A novel interaction between dynorphin(1–13) and an N-methyl-d-aspartate site

Dynorphin injected intrathecally in the rat results in a neurotoxicity behaviorally expressed as an irreversible loss of the thermally evoked tail-flick reflex. The excitatory amino acid antagonists DL-2-amino-5-phosphonovalerate (APV) and gamma-D-glutamylglycine (DGG) blocked the loss of the tail-flick reflex. The order of potency (APV greater than DGG) suggests that the N-methyl-D-aspartate (NMDA) subclass of excitatory amino acid receptors participate in the neurotoxicity. Additionally, intrathecal injection of APV results in a reversible loss of the tail-flick reflex, whereas with DGG doses which block the tail-flick reflex also result in hindlimb paralysis. These data suggest that neurotransmission in the tail-flick reflex pathway is, in part, mediated by NMDA receptors. From these and previous findings it was concluded that dynorphin neurotoxicity results from enhanced, excitotoxic, transmission across these synapses utilizing NMDA receptors.     

Results of N-methyl-D-aspartate antagonists in perinatal cerebral asphyxia therapy

Perinatal cerebral asphyxia, which results in significant neurologic and cognitive disabilities in infants and children, remains a major health problem. Potential neurologic sequelae include cerebral palsy, mental retardation, and epilepsy. Over the next few years, neuroprotective agents that prevent asphyxial neuronal injury and death are likely to be developed. These agents may also be effective in prophylaxis and treatment of chronic neurologic disorders, including epilepsy and neurodegenerative disorders, such as Huntington disease.     

Contribution of spinal N-Methyl-d-aspartic acid receptors to control of sympathetic outflow by the paraventricular nucleus

Spinal NMDA receptors contribute to control of the cardiovascular system by the ventrolateral medulla. However, little is known about the contribution of these receptors to suprabulbar regulation of hemodynamics and sympathetic outflow. Hence, the involvement of spinal NMDA receptors in regulation of the cardiovascular system by the paraventricular nucleus (PVN) of the hypothalamus was investigated. In urethane-anesthetized rats, the change in mean arterial pressure (MAP), heart rate (HR), and renal nerve activity (RNA) produced by electrical or chemical activation of the PVN was determined before and after intrathecal administration of the NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (AP5). Intrathecal AP5 decreased resting MAP, HR, and RNA, but had no effect on the increase in RNA produced by electrical or chemical stimulation of the PVN. The presser and renal vasoconstrictor effects resulting from electrical, but not chemical, stimulation of the PVN were significantly reduced by intrathecal AP5. These data show that much of the cardiovascular control exerted by the PVN does not depend on a spinal NMDA receptor mechanism.     

Dilatation of cerebral arterioles in response to N-methyl-d-aspartate: role of CGRP and acetylcholine

The purpose of these experiments was to examine mechanisms by which N-methyl-d-aspartate (NMDA) produces nitric oxide-dependent vasodilatation in brain. Some nitrovasodilators appear to dilate cerebral arterioles, in part, by release of calcitonin gene-related peptide (CGRP) from trigeminal fibers. The first goal of this study was to examine the hypothesis that dilatation of cerebral arterioles in response to NMDA is mediated by activation of receptors for CGRP. Diameters of cerebral arterioles were measured using a closed cranial window in anesthetized rabbits. Topical CGRP (1 and 10 nM) dilated cerebral arterioles by 30 ± 9 (mean±S.E.M.) and 72 ± 9%, respectively, from a control diameter of 94 ± 7 μm. This response was inhibited almost completely by the CGRP antagonist CGRP(8–37) (0.5 μM). NMDA (100 and 300 μM) dilated cerebral arterioles by 14 ± 5and38 ± 7% in the absence and 20 ± 5%and30 ± 6% in the presence, respectively, of CGRP(8–37). Neurons may release acetylcholine in response to activation with NMDA. The second goal of the present study was to examine the hypothesis that dilatation of cerebral arterioles in response to NMDA is mediated by acetylcholine. Topical atropine (2 μg/ml) completely inhibited dilatation of cerebral arterioles in response to acetylcholine, but had no effect on vasodilatation in response to NMDA. Thus, vasodilatation of cerebral arterioles in response to NMDA does not appear to be dependent on activation of receptors for CGRP or acetylcholine.     

Distribution of freed-serine in vertebrate brains

Freed-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, freed-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of freed-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio ofd-serine tol-serine, was d/l = 0.4), and low in the hindbrain. In developing mice,d-serine levels in the cerebrum increased with age and attained the adult level (d/l = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the freed-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: thed/l ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Freed-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain,d-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. Thed/l ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (d/l = 4), while that in the olfactory bulb was lower (d/l = 0.12). In the rat cerebrum, thed-serine content in the grey matter was significantly higher than that in the white matter. The contents of freed-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but thed/l ratio for bovine cerebral grey matter was lower than that for the cerebral white matter. Thed-serine level was discussed in terms ofd-amino-acid oxidase activity.     

Molecular and functional characterization of Xenopus laevis N-methyl-d-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors of many different vertebrates have been characterized in the past. However, little information is available about amphibian NMDA receptors. Here, we investigated the South African clawed frog Xenopus laevis NR1 subunit at the molecular and functional level. In this subunit, which is obligatory for functional NMDA receptor complexes, we found three exons, the N1, C1, and C3 cassettes, being alternatively spliced. Combinations of these cassettes generated six different splice variants, which were functionally characterized in oocytes. The Xenopus NR1 isoforms generally showed the same functional properties as their mammalian homologs when coexpressed with rat NR2B. In coexpression with Xenopus NR2B, however, some properties changed significantly. This included a Zn²⁺-mediated potentiation of current amplitudes for some subunit combinations which lasted for several minutes. This mechanism presents a novel form of Xenopus NMDA receptor modulation, possibly mediating a form of short-term potentiation in the Xenopus central nervous system.     

Blood-brain barrier permeability to leucine-enkephalin,d-Alanine-d-leucine-enkephalin and their N-terminal amino acid (tyrosine)

The permeability of the blood-brain barrier to [ tyrosyl-3,5-³H]enkephalin-(5-l-leucine) (abbreviated to Leu-Enk) and of its synthetic analogued-alanine²-[ tyrosyl-3,5-³H]enkephalin-(5-d-leucine) (abbreviated tod-Ala²-d-Leu⁵-Enk) was studied, in the adult rat, by means of Oldendorf's²⁷ intracarotid injection technique. The brain uptake index (BUI) corrected for residual vascular radioactivity was about the same for both peptides, indicating a low extraction from the blood during a 5- or 15-s period of exposure to the peptides. Transport of Leu-Enk was not saturated by unlabelled Enk at a concentration as high as 5 mM but was completely abolished by 5 mM tyrosine and by the inhibitor of aminopeptidase activity, bacitracin (2 mM). Also the typicall-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2 car☐ylic acid (BCH)⁹ at 10 mM concentration markedly reduced (by 80%) Leu-Enk uptake by the brain. In contrast, brain uptake ofd-Ala²-d-Leu⁵-Enk was reduced only to about one-half of its control value by bacitracin or by 25% by BCH. Brain uptake forl-tyrosine was typically large and markedly inhibited by BCH but not inhibited by 5 mM unlabelled Leu-Enk. These results show that the measurable but low first-pass extractions for enkephalins are not representative of the uptake of these peptides into the brain, but rather reflect their extreme sensitivity to enzymatic degradation with a release of the N-terminal tyrosine residue. The results also suggest that small amounts ofd-Ala²-d-Leu⁵-Enk might cross the blood-brain barrier in an intact form. It is concluded that the absence of a transport mechanism at the blood-brain barrier, together with a rapid enzymatic degradation, tends to prevent significant penetration of the barrier by Leu-Enk during the 5- or 15-s period of these studies.d-Ala²-d-Leu⁵-Enk however, shows a penetrance of the blood-brain barrier significantly greater than that of sucrose.     

Absence of side-effects in the anticonvulsant action of cortically applied antagonists of N-methyl-d-aspartate

Three compounds reportedly blocking the N-methyl-d-aspartate (NMDA) receptor, namely 2-amino-5-phosphonovalerate, γ-d-glutamylglycine and 3-hydroxy-2-quinoxalinecar☐ylic acid, were injected subdurally onto the cortex of freely moving rats. All 3 compounds effectively suppressed behavioral and electrographic seizure activity induced by strychnine, morphine and picrotoxin that were administered via the same route. The cortical application of the NMDA-receptor antagonists did not induce behavioral or electrographic changes, and behavioral side-effects commonly observed following intracerebroventricular administration of these compounds were absent. The anatomical separation of anticonvulsant action and side-effects induced by these compounds suggests that this class of compounds may eventually be useful as antiepileptic drugs.     

Oral administration of the oxidant-scavenger N-acetyl-l-cysteine inhibits acute experimental autoimmune encephalomyelitis

The prevention of acute experimental autoimmune encephalomyelitis (EAE) by N-acetyl-l-cysteine (NAC), a potent free radical scavenger, is described. Administrated ad libitum to SJL/J mice at a dosage of 0.2–2 mg/ml in drinking water from the day of the encephalitogenic injection, the agent significantly inhibited the induction of acute EAE. The improvement in clinical condition was dose-dependent. A complete protective effect required administration of the agent at an early stage. Examination of lymphocytes from NAC-treated EAE mice showed that at early stages (days 9 and 15) post encephalitogenic injection the anti-oxidant enhanced the specific lymphocyte proliferative response to the immunizing antigens. Examination of the mitogenic stimulation of lymphocytes from naive animals in the presence of NAC in vitro indicated that the scavenger enhanced the stimulative effect of LPS in a dose-dependent manner. The immunomodulative capacity of the anti-oxidant NAC suggests that free radicals are involved in the pathogenesis of acute EAE.     

Dose-dependent effects of d-N-allylnormetazocine on regional cerebral metabolic rates for glucose

Cerebral metabolic patterns produced by different doses of the benzomorphan opioid drug,d-N-allylnormetazocine (d-NANM), were stud using the 2-deoxy-d-[1-¹⁴C]glucose method in rats. The lowest dose ofd-NANM (0.5 mg/kg) decreased regional cerebral metabolic rates for glucose (rCMRglc) in areas, such as cranial nerve nuclei, that contain high densities of sigma (σ) receptors. However, higher doses of the drug (2.7 and 5 mg/kg) increased rCMRglc in components of the extrapyramidal motor and limbic systems. Some of these latter areas (e.g. molecular layer of the dentate gyrus, accumbens nucleus, globus pallidus, ventral posterior nucleus of the thalamus) are not enriched in σ receptors. Reductions in rCMRglc produced by the lowerst dose ofd-NANM probably reflect direct interactions of the drug with σ receptors, whereas increases in rCMRglc observed with the highest doses more likely result from effects ofd-NANM on PCP receptors.     

Release of [H]l-glutamine acid (l-Glu) and [H]d-aspartic acid (d-Asp) in the area of nucleus tractus solitarius in vivo produced by stimulation of the vagus nerve

We have investigated the effects of bilateral electrical stimulation of the vagus nerves in anesthetized, paralyzed rats on the release of exogenously administered [³H]l-glutamic acid ([³H]l-Glu) or [³H]d-aspartic acid ([³H]d-Asp) from the intermediate portion of the nucleus tractus solitarius (NTS). Electrical stimulation of afferent fibers with the frequency, pulse, duration, and intensity required to activate C-fibers, elicted hypotension and bradycardia. Such stimuli induced the release of [³H]l-Glu, or its stable analogue [³H]d-Asp, from the NTS into perfusate collected through push-pull cannulae. The release of radioactive materials, calculated as a percent of increase in radioactivity above the prestimulation level, was for [³H]l-Glu 114.4 ± 25.1% (n= 20) during bilateral vagal stimulation, and45.6 ± 11.3% (n= 9) (P < 0.001) during unilateral stimulation. The release of [³H]d-Asp induced by bilateral vagal stimulation was100.4 ± 31.9%. The release, which was anatomically specific and restricted to the NTS, was directly related to stimulus (and hence reflex) intensity. Overflow of the inert substances [¹⁴C]urea or [¹⁴C]sucrose, co-administered with the [³H]amino acids, did not increase at the same time. Local depolarization of the cells in the NTS by K⁺ (53 mM) increased the overflow of [³H]l-Glu, as well as [¹⁴C]urea, and was able to induce the release of [³H]l-Glu when electrical stimulation failed to have an effect. The results are consistent with the hypothesis thatl-Glu is a neurotransmitter of neurons in the NTS mediating vasodepressor response from vagal afferents, including those from systemic baroreceptors.     

Anomeric specificity ofd-glucose metabolism in rat brain cells

Rat brain cells were incubated at 9 °C (and occasionally at 37°C) in the presence of the anomers ofd-[5-³H]glucose,d-[U-¹⁴C]glucose,d-[6-¹⁴C]glucose andd-[1-¹⁴C]glucose (1.0 mM). The utilization of β-d-[5-³H]glucose was slightly higher than that of α-d-[5-³H]glucose, a situation possibly attributable to the anomeric behaviour of hexokinase. However, the production of¹⁴CO₂ from the α-anomer always largely exceeded that from the β-anomer. The anomeric difference ind-[U-¹⁴C]glucose oxidation, relative tod-[5-³H]glucose utilization, was suppressed in the presence of NH₄Cl. Even at anomeric equilibrium, the relative contribution of α-d-glucose to¹⁴CO₂ output exceeded its relative abundance. The β/α ratio ford-[1-¹⁴C]glucose oxidation (ord-[U-¹⁴C]glucose oxidation) was higher than that ford-[6-¹⁴C]glucose oxidation. Comparable observations were made in brain cells from albino rats and either lean or obese Zucker rats. It is concluded thatd-glucose metabolism displays anomeric specificity in rat brain cells, even when the latter are exposed to equilibratedd-glucose. It is also speculated that anomeric differences in the phosphorylation ofd-glucose by bound hexokinase may directly influence mitochondrial oxidative events.     

Simultaneous measurement of cholecystokinin- and vasoactive intestinal polypeptide-like immunoreactivity from cat frontal cortex in vitro: Effect of morphine andd-Ala-d-Leu-Enkephalin

The two peptides vasoactive intestinal polypeptide (VIP) and cholecystokinin (CCK) have been demonstrated to be discretely distributed in the cerebral cortex. This distribution closely parallels the distribution of μ- and δ-opiate receptors in the frontal cortex. The basal efflux and potassium-stimulated release of VIP- and CCK-immunoreactivity was studied in the presence and absence of morphine andd-Ala²-d-Leu⁵-enkephalin (DADL), agents with relative affinity for the μ and δ receptors, respectively. The basal efflux of VIP- and CCK-immunoreactivity was not affected by these opiate; however, the potassium-stimulated release of VIP-immunoreactivity was profoundly inhibited in a dose-dependent manner by both morphine (ED₅₀ = 1 × 10⁻⁹M) and DADL (ED₅₀= 3.02 × 10⁻⁹M). The inhibition produced by either morphine or DADL was shown to be reversed by naloxone.     

Contraversive circling induced by ventral tegmental microinjections of moderate doses of morphine and [d-Pen,d-Pen]enkephalin

Unilateral ventral tegmental area (VTA) injections of morphine and [d-Pen²,d-Pen⁵]enkephalin (DPDPE), caused contraversive circling at doses of 1.2, 12, and 24 nmol. Similar doses of the selective κ-agonist U-50,488H were ineffective. These data suggest a common mechanism for the circling, locomotion and facilitation of brain stimulation reward caused by VTA morphine, and distinguish this mechanism for that of feeding which is caused by both morphine and κ-actions in this region.     

Intrastriatal N-methyl-d-aspartate prevents amygdala kindled seizures in rats

Microinfusion of an excitatory amino acid, N-methyl-d-aspartate, into the ventral mid-striatum in the rat protects from amygdala kindled seizures. This result demonstrates that excitatory activity in the striatum modulates the threshold for seizures in the limbic forebrain.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.     

Transport of 2-deoxy-d-glucose by dissociated brain cells

The characteristics of glucose transport into dissociated cells from rat brain were determined using [1,2-³H]2-deoxyglucose as substrate. The rate of net uptake exhibited biphasic saturation kinetics with increasing substrate concentration; two values each for K_m (8.85 and 1.05 mM) and V_max (20.41 ± 5.99nmol/min/mg protein) were obtained, indicating the presence of two transport systems. d-glucose competed with [1,2-³H]2-deoxyglucose as shown by increasing degrees of inhibition of uptake of labeled substrate with increasing concentrations of d-glucose. The presence of an accelerative exchange mechanism was demonstrated by enhanced rates of uptake of labeled substrate by cells pre-loaded with high levels of unlabeled 2-deoxyglucose. Transport was inhibited by cytochalasin B, phloretin and phloridzin in a manner suggesting that the system is sodium-independent. Transport was also inhibited by sodium cyanide, potassium cyanide and dinitrophenol, but not by sodium arsenite or ouabain. Insulin status of the animals had no effect on the rate of transport of this substrate. Net transport was significantly lower in neonatal (4-day-old) rats than in either older sucklings (14–16-day-old) or adult animals; no significant difference between the latter two groups was observed. These findings demonstrate that two carrier-mediated systems for glucose transport are present on the membranes of these mixed brain cells suggesting that the kinetic characteristics of glucose transport may differ between neurons and glial cells. The age change in transport rate may reflect age-associated glial cell proliferation and/or an age-dependent increase in the number of transporters per cell in one brain cell type.     

Stimulation of tryptophan uptake into brain microvessels byd-glutamine

The uptake of L-tryptophan into isolated porcine microvessels is increased by preincubation with L-glutamine as well as with D-glutamine. This could indicate that gamma-glutamyltranspeptidase is involved in the stimulation of uptake of large neutral amino acids into the brain observed in hyperammonemic conditions.