Calcium-dependent effects of melatonin inhibition of glutamatergic response in rat striatum

The effects of melatonin, amlodipine, diltiazem (L-type Ca2+ channel blockers) and omega-conotoxin (N-type Ca2+ channel blocker) on the glutamate-dependent excitatory response of striatal neurones to sensory-motor cortex stimulation was studied in a total of 111 neurones. Iontophoresis of melatonin produced a significant attenuation of the excitatory response in 85.2% of the neurones with a latency period of 2 min. Iontophoresis of either L- or N-type Ca2+ channel blocker also produced a significant attenuation of the excitatory response in more than 50% of the recorded neurones without significant latency. The simultaneous iontophoresis of melatonin + amlodipine or melatonin + diltiazem did not increase the attenuation produced by melatonin alone. However, the attenuation of the excitatory response was significantly higher after ejecting melatonin + omega-conotoxin than after ejecting melatonin alone. The melatonin-Ca2+ relationship was further supported by iontophoresis of the Ca2+ ionophore A-23187, which suppressed the inhibitory effect of either melatonin or Ca2+ antagonists. In addition, in synaptosomes prepared from rat striatum, melatonin produced a decrease in the Ca2+ influx measured by Fura-2AM fluorescence. Binding experiments with [3H]MK-801 in membrane preparations from rat striatum showed that melatonin did not compete with the MK-801 binding sites themselves although, in the presence of Mg2+, melatonin increased the affinity of MK-801. The results suggest that decreased Ca2+ influx is involved in the inhibitory effects of melatonin on the glutamatergic activity of rat striatum.     

N-methyl-D-aspartate receptor subunit phenotypes of vagal afferent neurons in nodose ganglia of the rat

Most vagal afferent neurons in rat nodose ganglia express mRNA coding for the NR1 subunit of the heteromeric N-methyl-D-aspartate (NMDA) receptor ion channel. NMDA receptor subunit immunoreactivity has been detected on axon terminals of vagal afferents in the dorsal hindbrain, suggesting a role for presynaptic NMDA receptors in viscerosensory function. Although NMDA receptor subunits (NR1, NR2B, NR2C, and NR2D) have been linked to distinct neuronal populations in the brain, the NMDA receptor subunit phenotype of vagal afferent neurons has not been determined. Therefore, we examined NMDA receptor subunit (NR1, NR2B, NR2C, and NR2D) immunoreactivity in vagal afferent neurons. We found that, although the left nodose contained significantly more neurons (7,603), than the right (5,978), the proportions of NMDA subunits expressed in the left and right nodose ganglia were not significantly different. Immunoreactivity for NMDA NR1 subunit was present in 92.3% of all nodose neurons. NR2B immunoreactivity was present in 56.7% of neurons; NR2C-expressing nodose neurons made up 49.4% of the total population; NR2D subunit immunoreactivity was observed in just 13.5% of all nodose neurons. Double labeling revealed that 30.2% of nodose neurons expressed immunoreactivity to both NR2B and NR2C, whereas NR2B and NR2D immunoreactivities were colocalized in 11.5% of nodose neurons. NR2C immunoreactivity colocalized with NR2D in 13.1% of nodose neurons. Our results indicate that most vagal afferent neurons express NMDA receptor ion channels composed of NR1, NR2B, and NR2C subunits and that a minority phenotype that expresses NR2D also expresses NR1, NR2B, and NR2C.     

Influence of maternal melatonin on melatonin receptors in rat offspring

Summary Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis.     

Endoplasmic reticulum-associated degradation of the NR1 but not the NR2 subunits of the N-methyl-D-aspartate receptor induced by inhibition of the N-glycosylation in cortical neurons

The N-methyl-D-aspartate receptor (NMDAR) is fundamental to normal and pathological functioning of neurons. The receptor subunits are N-glycosylated proteins synthesized in the endoplasmic reticulum (ER) that fold, mature, and oligomerize as they transit through the secretory pathway. Although the early processes of biogenesis are fundamental to NMDAR expression and function, our knowledge of them is nevertheless limited. Additionally, the investigation of NMDAR synthesis is highly relevant, in that ER dysfunction, frequently associated with acute and degenerative brain diseases, might alter this process. We characterize here the effect of ER stress produced by inhibition of N-glycosylation on NMDAR synthesis and function. We use first heterologous systems of NMDAR expression in which NR1 and NR2A subunits are synthesized in nonneuronal cells. The function of these NMDARs as Ca2+ channels is repressed by tunicamycin, because of the inhibition of NR1, but no NR2A, synthesis. The regulation of NR1 is relevant to the central nervous system, in that a dramatic decrease in synthesis of this subunit and assembly of NMDARs is observed in cortical neurons treated with tunicamycin. The inhibition of NR1 synthesis is not due to changes in levels of mRNA but associated with the earliest stages in NMDAR biogenesis. The inhibition of N-glycosylation activates ER-specific stress responses in neurons, which include the ER-associated degradation (ERAD) mechanism responsible for differential and extremely efficient degradation of nonglycosylated NR1 by the proteasome after ubiquitination. Because this is an obligatory NMDAR component, the significant sensitivity of NR1 to ER stress will have important consequences on receptor function.     

Inner retinal neurons display differential responses to N-methyl-D-aspartate receptor activation

The N-methyl-D-aspartate (NMDA) responses of neurons from within the inner rabbit retina were mapped using a channel permeable cation, 1-amino-4-guanidobutane (agmatine, AGB). Serial sections were subsequently probed with immunoglobulins targeting AGB, glutamate, gamma-aminobutyric acid (GABA), and glycine to visualize the NMDA responses of neurochemical subpopulations of neurons. Most inner retinal subpopulations of neurons demonstrated an NMDA concentration-dependent increase in activation. This NMDA-induced activation displayed a distinct pattern, with the most sensitive class to least sensitive class ranking being GC > GABA cAC > GABA/Gly cAC > Gly cAC > GABA dAC (GC, ganglion cells; AC, amacrine cells; c, conventional; d, displaced; Gly, glycine). The variable NMDA response may reflect differences in NMDA receptor subunit disposition or differences in receptor density. In addition to the variable NMDA activation pattern, we found that virtually all ganglion cells (87%) showed NMDA-gated AGB entry, compared with only 58% of amacrine cells. We conclude that a large cohort of amacrine cells do not possess functional NMDA receptors. In addition to most ganglion cells being activated by NMDA, a large subpopulation displayed the highest sensitivity to NMDA application. The functional significance of this finding is that the ganglion cell population will be the first neuronal class to be susceptible to glutamate-induced neurotoxicity mediated through the NMDA receptor. The addition of betaxolol significantly reduced NMDA-mediated AGB entry into most neuronal groups (ganglion cells, GABA, and glycine amacrine cells), with the greatest effect being on ganglion cells. Betaxolol had no significant effect on NMDA-gated entry of AGB on the GABA/Gly amacrine cell population.     

Effects of chronic melatonin administration on GABA and diazepam binding in rat brain

Summary In order to determine whether the in vitro ability of the pineal indoleamine hormone, melatonin, to modulate binding at the GABA-benzodiazepine receptor complex is operative in vivo we have examined the effects of chronic melatonin administration on³H-GABA and³H-diazepam binding in rat brain.Melatonin was injected daily in increasing doses for three weeks and animals were sacrificed at 2 or 26 hours after the final injection. A melatonin-induced enhancement of³H-GABA binding was observed in both single-point and saturation binding experiments.Scatchard analysis of³H-diazepam binding revealed a melatonin-induced increase in binding affinity at 26 hours in the forebrain and at 2 hours in the cerebellum with no significant changes in binding site concentration.These findings are consistent with the proposal that melatonin's psychopharmacological effects are due at least in part to its ability to enhance central GABAergic transmission by modulating GABA receptor activity.     

Mechanisms of NMDA receptor inhibition by diarylamidine compounds

Pentamidine, diminazene and 4′,6‐diamidino‐2‐phenylindole (DAPI) are antiprotozoal diarylamidine compounds. In the present work, we have studied their action on native N‐methyl‐D‐aspartate (NMDA) receptors in rat hippocampal pyramidal neurons. All three compounds inhibited NMDA receptors at ?80 mV holding voltage with IC₅₀ of 0.41 ± 0.08, 13 ± 3 and 3.1 ± 0.6 μM, respectively. The inhibition by pentamidine was strongly voltage‐dependent, while that of DAPI was practically voltage‐independent. Inhibition by diminazene had both voltage‐dependent and voltage‐independent components. Diminazene and DAPI demonstrated tail currents and overshoots suggesting “foot‐in‐the‐door” mechanism of action. In contrast, pentamidine was partially trapped in the closed NMDA receptor channels. Such difference in the mechanism of action can be explained by the difference in the 3D structure of compounds. In the pentamidine molecule, two benzamidine groups are connected with a flexible linker, which allows the molecule to fold up and fit in the cavity of a closed NMDA receptor channel. Diminazene and DAPI, in contrast, have an extended form and could not be trapped.     

Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography

The N-methyl-D-aspartate (NMDA) receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In this study, an effect of age on NMDA antagonist binding sites was investigated through quantitative in vitro autoradiography with 3H-3-((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). 3H-CPP binding sites were most concentrated in the hippocampus and cerebral cortex where NMDA receptors have been demonstrated to be involved in synaptic transmission. In aged rats, 3H-CPP binding sites in the hippocampus and cerebral cortex were not significantly changed. As for other brain regions, there was an age-dependent decline of binding sites only in the caudate-putamen and nucleus accumbens. Our previous study revealed that strychnine-insensitive glycine receptors were markedly reduced in telencephalic regions in the aged rat brain. Taking these findings into consideration, it is concluded that glycine receptors but not NMDA antagonist binding sites are severely altered in telencephalic regions of aged animals. It is considered that within the NMDA receptor complex, glycine receptors may be selectively affected in the aging process.     

Modulation of glutamate receptor expression by gonadal steroid hormones in the rat striatum

The non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) attenuates morphine-induced immediate-early gene expression in the rat striatum in a sexually dimorphic manner that depends in part on gonadal steroids. To determine if this effect was dependent on modulation of glutamate receptor gene expression, we studied the effect of gonadal hormones on levels of the NR1 subunit of NMDA receptor and the GluR2 subunit of the AMPA-subtype of glutamate receptor in the rat striatum, using autoradiographic immunocytochemistry. We found that ovariectomy decreased GluR2 immunoreactivity in the striatum, but no changes were seen in levels of NR1 following gonadectomy in either sex. Thus, the effects of gonadal steroids on NMDA receptor-mediated responses are not due to regulation of NR1 expression.     

3-Hydroxykynurenine potentiates quinolinate but not NMDA toxicity in the rat striatum

L-3-Hydroxykynurenine (L-3-HK) and quinolinate (QUIN) are two metabolites of the kynurenine pathway, the major route of tryptophan degradation in mammals. L-3-HK is a known generator of highly reactive free radicals, whereas QUIN is an endogenous excitotoxin acting specifically at N-methyl-D-aspartate (NMDA) receptors. This study was designed to examine possible synergistic interactions between L-3-HK and QUIN in the rat brain in vivo. Intrastriatal coinjection of 5 nmol L-3-HK and 15 nmol QUIN, i.e. doses which caused no or minimal neurodegeneration on their own, resulted in substantial neuronal loss, determined both behaviourally (apomorphine-induced rotations) and histologically (quantitative assessment of lesion size). The excitotoxic nature of the lesion was verified by tyrosine hydroxylase immunohistochemistry, showing the survival of dopaminergic striatal afferents. There was also a relative sparing of large striatal neurons, and neurodegeneration was prevented both by NMDA receptor blockade (using CGP 40116) and free radical scavenging [using N-tert-butyl-alpha-(2-sulphophenyl)-nitrone, S-PBN]. The pro-excitotoxic features of L-3-HK were especially pronounced at low QUIN doses and were not observed when QUIN was substituted by NMDA. Notably, the effect of L-3-HK was not due to its intracerebral conversion to QUIN and was duplicated by equimolar D,L-3-HK. These data indicate that an elevation of L-3-HK levels constitutes a significant hazard in situations of excitotoxic injury. Pharmacological interventions aimed at decreasing L-3-HK formation may therefore be particularly useful for the treatment of neurological diseases which are associated with an abnormally enhanced flux through the kynurenine pathway.     

Effect of antipsychotic drugs on brain-derived neurotrophic factor expression under reduced N-methyl-D-aspartate receptor activity

Brain-derived neurotrophic factor (BDNF) promotes a variety of neuromodulatory processes during development as well as in adulthood. This neurotrophin has been associated with synaptic plasticity, suggesting that its regulation may represent one of the mechanisms through which psychotropic drugs alter brain function. Because reduced glutamatergic function represents a major feature of schizophrenia, we investigated the effects of the concomitant administration of haloperidol or olanzapine with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on BDNF expression. MK-801 reduces the hippocampal expression of the neurotrophin; this effect was exacerbated by haloperidol, but it was normalized by olanzapine. Our data reveal a fine tuning of BDNF biosynthesis and a differential modulation by antipsychotic drugs when NMDA-mediated transmission is reduced, suggesting that haloperidol and olanzapine can produce different effects on brain plasticity through the modulation of BDNF expression.     

Effect of delta-aminolevulinic acid and vitamin E treatments on the N-methyl-D-aspartate receptor at different ages in the striatum of rat brain

We report the effects of the chronic treatments with the oxidant agent delta-aminolevulinic acid (ALA) and with the antioxidant vitamin E on the N-methyl-D-aspartate (NMDA) receptors in the striatum of 4-, 12- and 24-month-old male Wistar rats. ALA and vitamin E were administered daily for 15 days (40 mg/kg i.p. and 20 mg/kg i.p. respectively). NMDA receptors were labeled by membrane homogenate binding, using tritiated dizocilpine ([3H]MK-801). [3H]MK-801 binding in the striatum was significantly decreased at all ages in ALA-treated rats with respect to their controls, and in contrast, was significantly increased at all ages when rats received the treatment with vitamin E. Western blot assays were performed using antibodies against the NR2A subunit, a NMDA receptor subunit widely distributed in the brain. We did not find significant differences in the amounts of NR2A in rats treated with either ALA or vitamin E with respect to those rats not treated. We conclude that the NMDA receptor densities in the rat striatum are modified by the chronic treatment with oxidants and antioxidants in an age-independent way, at least until 24 months. Also, our results support the notion that NR2A is not involved in these modifications.     

Circadian photic regulation of melatonin receptor density in rat suprachiasmatic nuclei: Comparison with light induction of fos-related protein

High-affinity melatonin receptors are present in rat suprachiasmatic nuclei (SCN), and their density exhibits a daily rhythm regulated by the light/dark cycle. In this study we demonstrate that the light regulation of these receptors depends on a circadian mechanism. Pinealectomized rats kept in constant darkness were subjected to 1-hr light pulses delivered across the circadian cycle. The density of melatonin receptors was significantly increased when photic exposure was performed during subjective night, and not different from control animals kept in darkness when the light pulse was applied during subjective day. The protein product (Fos) of the immediate early gene c-fos studied in the same paradigm showed globally the same circadian sensitivity phase. These results clearly show that, although the rhythmic appearance of melatonin receptor density in SCN follows and is directly regulated by the standard light/dark cycle, this light regulation is not passive. As is the case with Fos-like protein, it is only during a precise phase of the circadian cycle that light is able to regulate the density of melatonin receptors in SCN. © 1996 Wiley-Liss,Inc.     

Somatostatin receptor elevation in rat striatum after diisopropylfluorophosphate administration

The acute and chronic administration of diisopropylfluorophosphate (DFP), an inhibitor of acetylcholinesterase or of atropine, a blocker of muscarinic cholinergic receptors, did not affect somatostatin-like immunoreactivity (SLI) content in the striatum of rats. Acute and chronic DFP administration increased the number of specific 125I-Tyr11-somatostatin (125I-Tyr11-SS) receptors in cells dissociated from the striatum without changing the affinity constant. Although the increase could be blocked by pretreatment with atropine, it was not due to a direct effect by DFP on somatostatin (SS) receptors, because no rise in 125I-Tyr11-SS binding was produced by high concentrations of DFP (10(-5) M) when added in vitro. The acute administration of atropine alone had no observable effect on the number of SS receptors. However, repeated atropine administration produced a significant decrease in the 125I-Tyr11-SS binding in cells dissociated from the striatum, although the affinity constant was unchanged. The results suggest that interactions between somatostatinergic and cholinergic receptors may be of importance in the rat striatum.     

Distinct expression of phosphorylated N-methyl-D-aspartate receptor NR1 subunits by projection neurons and interneurons in the striatum of normal and amphetamine-treated rats

N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of subunits (NR1 and NR2A-D), and are enriched in the striatum. Receptor phosphorylation has recently been demonstrated on the NR1 subunit at three serine residues, 897, 896, and 890, which appear to correspond to the level of receptor activity. In this study, expression of phospho-specific NR1 subunits at serine 897 (pNR1S897), serine 896 (pNR1S896), or serine 890 (pNR1S890) in neurochemically identified neurons of the adult rat striatum was detected by using double-immunofluorescent labeling or combined in situ hybridization and immunohistochemistry. In both the dorsal and ventral striatum, pNR1S897 was expressed at high levels in projection neurons containing >55% dynorphin (striatonigral) and >90% enkephalin (striatopallidal) and in interneurons that were 100% positive for choline, >90% positive for parvalbumin, and >45% positive for somatostatin (co-containing neuropeptide Y and neuronal nitric oxide synthase). Low levels of pNR1S896 were present in a small portion of projection neurons (<15% for both populations of projection neurons) and were almost lacking in the three types of interneurons. Interestingly, pNR1S890 was exclusively expressed in most parvalbumin-containing interneurons (70-80%). Acute administration of a psychostimulant, amphetamine, increased the number of dynorphin-containing projection neurons and parvalbumin interneurons showing detectable levels of pNR1S896 and pNR1S890, respectively. These results demonstrate the distinct expression of phospho-NR1 subunits in different populations of striatal projection neurons and interneurons at variable levels in normal rats; they also demonstrate that phosphorylation of NR1, at least on serine 896 and 890 sites, is sensitive to drug exposure.     

Chronic ethanol changes opiate receptor function in rat striatum

Ethanol produces supersensitivity of striatal σ-opiate receptor sites labeled by [³H]DADLE and [³H]etorphine. The impairment may be ascribed to the diminished enkephalin release detected in rat striatum after chronic ethanol consumption. On the contrary, a lower affinity of striatal μ-opiate receptors results after same ethanol exposure. In fact, the K_d values of [³H]Met-enkephalin and [³H]DHM are enhanced when measured in striata of ethanol-dependent rats. The diverse sensitivity of the various classes of opiate receptors to ethanol may be ascribed to different ethanol effects on enekephalinergic transmission.     

Antisense oligonucleotides to N-methyl-D-aspartate receptor subunits attenuate formalin-induced nociception in the rat

Noxious peripheral stimuli increase the sensitivity of central nociceptive neurons to subsequent noxious stimuli. This occurs in part through activation of spinal N-methyl-D-aspartate (NMDA) receptors. These receptors are heteromeric complexes of NMDA-R1 and NMDA-R2 A--D subunits. NMDA-R1 is necessary for the formation of functional NMDA receptors whereas the R2 subunits (A-D) modify the properties of the receptor. However, the role of the various receptor subtypes in nociception has not been established. In this study, we used intrathecally administered phosphodiester antisense oligonucleotides (ODEs) to examine the role of the NMDA-R1, NMDA-R2C and NMDA-R2D subunits in the mediation of formalin-induced nociception in the rat. The antisense ODEs against the NMDA-R1 and NMDAR-2C subunits reduced nociceptive behaviors whereas the corresponding sense ODEs had no effect. In contrast, nociception was unaffected by the antisense ODE to NMDAR-2D. Using an RNase protection assay, we also found that each antisense ODE selectively decreased the level of the corresponding mRNA in the lumbar spinal cord but that the sense ODEs had no such effect. Accordingly, these data provide evidence that the R1 and R2C subunits, but not R2D, of the NMDA receptor participate in the development of formalin-induced nociception.     

海马NMDA受体调节严重烫伤应激后HPA轴兴奋性的相关机制研究

目的探寻海马N-甲基-D-天冬氨酸(NMDA)受体调节严重创伤应激后HPA轴过度兴奋的可能机制。方法以30%总体表面积(TBSA)Ⅲ度烫伤应激作为严重创伤应激模型,先通过地塞米松抑制试验检测严重烫伤应激后糖皮质激素(GC)负反馈功能的变化,再利用RT-PCR技术检测烫伤应激后海马糖皮质激素受体(GR)mRNA水平(其水平与负反馈功能密切相关)的变化特点,并观察烫伤应激前腹腔注射NMDA受体拮抗剂MK-801对烫伤应激后2hGRmRNA水平的影响。结果30%TBSAⅢ度烫伤应激后地塞米松抑制试验阴性,GC负反馈功能下降;烫伤应激后0.5、2、8、24、48h海马GRmRNA水平皆明显降低,尤以伤后2h最明显;与烫伤应激组相比,MK-8013mg/kg组GRmRNA水平显著上升,MK-8016mg/kg组海马GRmRNA水平进一步上升,盐水组GRmRNA水平无明显变化。结论海马NMDA受体调节严重烫伤应激后HPA轴的亢进是通过下调海马GR从而影响了GC在海马水平的负反馈引起的。