Nicotine improves morphine-induced impairment of memory: possible involvement of N-methyl-D-aspartate receptors in the nucleus accumbens

The possible involvement of N-methyl-D-aspartate (NMDA) receptors in the nucleus accumbens (NAc) in nicotine's effect on impairment of memory by morphine was investigated. A passive avoidance task was used for memory assessment in male Wistar rats. Subcutaneous (s.c.) administration of morphine (5 and 10 mg/kg) after training impaired memory performance in the animals when tested 24 h later. Pretest administration of the same doses of morphine reversed impairment of memory because of post-training administration of the opioid. Moreover, administration of nicotine (0.2 and 0.4 mg/kg, s.c.) before the test prevented impairment of memory by morphine (5 mg/kg) given after training. Impairment of memory performance in the animals because of post-training administration of morphine (5 mg/kg) was also prevented by pretest administration of a noncompetitive NMDA receptor antagonist, MK-801 (0.75 and 1 microg/rat). Interestingly, an ineffective dose of MK-801 (0.5 microg/rat) in combination with low doses (0.075 and 0.1 mg/kg) of nicotine, which had no effects alone, synergistically improved memory performance impaired by morphine given after training. On the other hand, pretest administration of NMDA (0.1 and 0.5 microg/rat), which had no effect alone, in combination with an effective dose (0.4 mg/kg, s.c.) of nicotine prevented the improving effect of nicotine on memory impaired by pretreatment morphine. The results suggest a possible role for NMDA receptors of the NAc in the improving effect of nicotine on the morphine-induced amnesia.     

Activation of metabotropic glutamate receptors in conjunction with postsynaptic depolarization triggers a long-term depression of the N-methyl-D-aspartate receptor-mediated synaptic potential in the rat hippocampus

The mechanism responsible for long-term depression (LTD) of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic potential (EPSP_NMDA) was studied. Intracellular recordings were made from CA1 cells of rat hippocampal slices in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) and picrotoxin (50 µM), which block non-NMDA and GABA_A receptors, respectively. Intracellular injections of depolarizing pulses (500 ms, 0.3–0.7 nA) at 1 Hz for 5 min in the absence of synaptic stimulation caused a persistent increase in the amplitude of EPSP_NMDA. However, coupling postsynaptic depolarization with synaptic activity induced LTD. The EPSP_NMDA LTD could be blocked byL-2-amino-3-phosphonopropionic acid (50 µM) or (RS)--methyl-4-carboxyphenylglycine (200 µM), specific antagonists for metabotropic glutamate receptors (mGluR). Furthermore, application oftrans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 µM), a specific mGluR agonist, in conjunction with postsynaptic depolarizing elicited LTD. In contrast, the mGluR agonists quisqualate or t-ACPD when given alone produced a sustained enhancement of EPSP_NMDA. Finally, coupled depolarization did not evoke LTD in slices pretreated with the protein kinase C (PKC) inhibitor calphostin c (60 nM). The present results demonstrate that activation of mGluR is necessary for the induction of LTD of EPSP_NMDA and suggest that NMDA receptors are subject to bidirectional regulation by mGluR. Furthermore, the induction of LTD is likely to involve the stimulation of PKC.     

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased contribution of N-methyl-D-aspartate receptors to synaptic transmission inCA1 area of the rat hippocampus after short-term episodes of hypoxia/aglycemia

Effect of hypoxia/aglycemia episodes on excitatory postsynaptic currents (EPSC) evoked in pyramidal neurons of the rat hippocampalCA1 area by electrical stimulation of Schaffer collaterals was studied using voltage-clamp and intracellular perfusion techniques. By 60–80 min after a 10-min-long hypoxia/aglycemia episode, the EPSC amplitude increased and the EPSC decay was considerably slowed down, if compared with control. In contrast to control conditions, under which EPSC decay kinetics did not depend on the stimulus strength, hypoxia/aglycemia was followed by slowing down of the EPSC decay when stimulus intensity increased. The stimulus-dependent posthypoxic “slow” EPSC component was depressed both by D-(−)-2-amino-5-phosphonovaleric acid, an NMDA receptor blocker, and by 6-cyano-7-nitroquinoline-2,3-dion, a non-NMDA receptor blocker, which suggested possible polysynaptic origin of the above EPSC component. We suggest that short-term hypoxia/aglycemia transforms into an active state the NMDA receptors in the synapses of excitatory reccurrent collaterals of theCA1 hippocampal area, which had not functioned before. An increase in the intracellular calcium concentration from 1.5 to 5.0 mM resulted in the effect similar to that produced by hypoxia/aglycemia, which suggests that calcium channels play an important role in the mechanisms responsible for hypoxia-related activation of “silent” NMDA receptors.     

Vinpocetine prevents ischemic cell damage in rat hippocampus

The effects of vinpocetine on hippocampal cell damage and local cerebral blood flow (LCBF) were measured in a rat model of forebrain ischemia (2-vessel occlusion and hypotension). Duration of ischemia was 10 min. LCBF was determined after 2 min of recirculation using the 14C-iodoantipyrine technique. Hippocampal cell loss was quantified histologically 7 days post-ischemia. Intraperitoneal application of vinpocetine (10 mg/kg) 15 min prior to ischemia significantly reduced neuronal cell loss in hippocampal CA 1 sector from 60% to 28%. The drug led to a marked increase in blood flow in cortical areas, whereas LCBF remained unchanged in hippocampus and all other structures measured. It is suggested that the protective effect of vinpocetine does not depend on increased postischemic blood flow.     

In vivo activation of N-methyl-D-aspartate receptors in the rat hippocampus increases prostaglandin E(2) extracellular levels and triggers lipid peroxidation through cyclooxygenase-mediated mechanisms

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE(2) and 8-epi-PGF(2alpha) following infusion of N-methyl-D-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE(2) and 8-epi-PGF(2alpha) basal levels. These effects were largely prevented by the specific cytosolic phospholipase A(2) (cPLA(2) ) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.     

Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity

The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [³H]MK-801 labeled a uniform population of sites (K_d = 3.2 ± 0.3 nM) in a saturable manner (B_max = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [³H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [³H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.     

Modulation of ATP-induced LTP by cannabinoid receptors in rat hippocampus

Cannabinoids exert powerful action on various forms of synaptic plasticity. These retrograde messengers modulate GABA and glutamate release from presynaptic terminals by acting on presynaptic CB1 receptors. In particular, they inhibit long-term potentiation (LTP) elicited by electrical stimulation of excitatory pathways in rat hippocampus. Recently, LTP of the field excitatory postsynaptic potential (fEPSP) induced by exogenous ATP has been thoroughly explored. The present study demonstrates that cannabinoids inhibit ATP-induced LTP in hippocampal slices of rat. Administration of 10 μM of ATP led to strong inhibition of fEPSPs in CA1/CA3 hippocampal synapses. Within 40 min after ATP removal from bath solution, robust LTP was observed (fEPSP amplitude comprised 130.1 ± 3.8% of control, n = 10). This LTP never appeared when ATP was applied in addition to cannabinoid receptor agonist WIN55,212-2 (100 nM). Selective CB1 receptor antagonist, AM251 (500 nM), completely abolished this effect of WIN55,212-2. Our data indicate that like canonical LTP elicited by electrical stimulation, ATP-induced LTP is under control of CB1 receptors.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9296-5) contains supplementary material, which is available to authorized users.     

Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine ( l -CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3-pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3-nitropropionic acid (3-NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with l -CAR, chronically injected with 3-NP and/or intrastriatally infused with QUIN. At micromolar concentrations, l -CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. l -CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3-NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein-immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3-NP) were reduced by l -CAR. In addition, l -CAR prevented the synergistic action of 3-NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of l -CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.     

In vivo hypoxia-induced neuronal damage in dentate gyrus of rat hippocampus: changes in NMDA receptors and the effect of MK–801

Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O₂ and 95% N₂ for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [³H]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate (MK–801), on against in vivo hypoxia. The administration of MK–801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR.     

Novel adenosine receptors in rat hippocampus. Identification and characterization

2-Chloro[3H]adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a KD of 4.7 microM and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability.     

Cyclo-oxygenase-1 and -2 differently contribute to prostaglandin E2 synthesis and lipid peroxidation after in vivo activation of N-methyl-D-aspartate receptors in rat hippocampus

Using intracerebral microdialysis, we reported previously that acute in vivo activation of NMDA glutamate receptors triggers rapid and transient releases of prostaglandin E2 (PGE2) and F2-isoprostane 15-F(2t)-IsoP in the hippocampus of freely moving rats. The formation of the two metabolites--produced through cyclo-oxygenase (COX) enzymatic activity and free radical-mediated peroxidation of arachidonic acid (AA), respectively,--was prevented by the specific NMDA antagonist MK-801, and was largely dependent on COX-2 activity. Here, we demonstrate that besides COX-2, which is the prominent COX isoform in the brain and particularly in the hippocampus, the constitutive isoform, COX-1 also contributes to prostaglandin (PG) synthesis and oxidative damage following in vivo acute activation of hippocampal NMDA glutamate receptors. The relative contribution of the two isoforms is dynamically regulated, as the COX-2 selective inhibitor NS398 immediately prevented PGE2 and 15-F(2t)-IsoP formation during the application of NMDA, whereas the COX-1 selective inhibitor SC560 was effective only 1 h after agonist infusion. Our data suggest that, although COX-2 is the prominent isoform, COX-1 activity may significantly contribute to excitotoxicity, particularly when considering the amount of lipid peroxidation associated with its catalytic cycle. We suggest that both isoforms should be considered as possible therapeutic targets to prevent brain damage caused by excitotoxicity.     

Adenosine A2A receptors and metabotropic glutamate 5 receptors are co-localized and functionally interact in the hippocampus: a possible key mechanism in the modulation of N-methyl-D-aspartate effects

Hippocampal metabotropic glutamate 5 receptors (mGlu5Rs) regulate both physiological and pathological responses to glutamate. Because mGlu5R activation enhances NMDA-mediated effects, and given the role played by NMDA receptors in synaptic plasticity and excitotoxicity, modulating mGlu5R may influence both the physiological and the pathological effects elicited by NMDA receptor stimulation. We evaluated whether adenosine A2A receptors (A(2A)Rs) modulated mGlu5R-dependent effects in the hippocampus, as they do in the striatum. Co-application of the A(2A)R agonist CGS 21680 with the mGlu5R agonist (RS)-2-chloro-s-hydroxyphenylglycine(CHPG) synergistically reduced field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices. Endogenous tone at A(2A)Rs seemed to be required to enable mGlu5R-mediated effects, as the ability of CHPG to potentiate NMDA effects was antagonized by the selective A(2A)R antagonist ZM 241385 in rat hippocampal slices and cultured hippocampal neurons, and abolished in the hippocampus of A(2A)R knockout mice. Evidence for the interaction between A(2A)Rs and mGlu5Rs was further strengthened by demonstrating their co-localization in hippocampal synapses. This is the first evidence showing that hippocampal A(2A)Rs and mGlu5Rs are co-located and act synergistically, and that A(2A)Rs play a permissive role in mGlu5R receptor-mediated potentiation of NMDA effects in the hippocampus.     

学习训练对谷氨酸单钠所致大鼠海马损伤的神经保护作用

目的：探讨学习训练对谷氨酸神经毒性的保护作用。方法：在SD大鼠生后第3～9d腹腔注射谷氨酸单钠复制谷氨酸毒性模型,在1月龄和2月龄时训练大鼠学会以明暗辨别来获得食物,3月龄时取脑,在光镜下计数海马内存活神经元数,电镜下观察海马CA1区的超微结构,并计数突触数,测量突触活性带长度。结果：学习训练组海马CA3区和CA4区内的存活神经元数、海马CA1区内的突触数和突触活性带长度均大于非学习组,结论：结果提示学习训练可在一定程度上减轻MSG对海马的损伤。     

Binding of corticosteroid receptors to rat hippocampus nuclear matrix.

In rat hippocampus, the mineralocorticoid receptor and the glucocorticoid receptor bind corticosterone with high affinity. We have studied the association of these receptors with the nuclear matrix both after in vivo and in vitro administration of radiolabelled corticosterone to hippocampus cells. It was found that in vivo 100% and in vitro 60% of the corticosterone that specifically bound to rat hippocampus nuclei was attached to the nuclear matrix. A selective glucocorticoid receptor agonist did not compete for corticosterone binding. This indicates that this binding was mediated by the mineralocorticoid receptor rather than the glucocorticoid receptor.     

Comparative mapping of opioid receptors and enkephalin immunoreactive nerve terminals in the rat hippocampus

Summary Opioid receptors can be localized to the hippocampal formation of the rat by autoradiography. The binding of ³H-enkephalinamide to fixed and mounted tissue sections has all the characteristics associated with binding to opioid receptors. It is saturable, of high affinity and displays stereospecificity. The opioid receptor distribution shows striking regional variation throughout the hippocampal formation. Areas with high density include the pyramidal cell layer of both regio superior (CA1) and regio inferior (CA3), stratum moleculare of the hippocampus, the cell layer of subiculum, the superficial part of presubiculum and the deep layer (VI) of the medial and lateral entorhinal cortices. Areas with low to medium densities include regions corresponding to the dendritic field of the pyramidal cells (str. oriens, str. radiatum and the mossy fiber zone), the dentate granule cell layer and the molecular layer of the dentate area. Enkephalin-like immunoreactivity is detected in both intrinsic neuronal systems: 1) the mossy fibers which terminate on the proximal part of the CA3 pyramidal cell dendrites and on CA4 pyramidal cells, 2) cell bodies with multiple short processes, probably interneurons, dispersed throughout the hilus of the dentate area, the pyramidal cell layer of hippocampus, the str. radiatum, and occasionally in the str. moleculare and in the str. oriens, and extrinsic neuronal systems: 1) the lateral perforant path and 2) the lateral temporo-ammonic tract. Thus, the hippocampus contains intrinsic systems of enkephalin-like immunoreactive nerve terminals which may exert their effect on the opioid receptors with a localization corresponding to the pyramidal cells and their apical dendrites. Extrinsic enkephalinergic systems corresponding to the terminal fields of the lateral perforant path and the temporoammonic tract, both of entorhinal origin, may influence the opioid receptors located in the molecular layer of the dentate area, and in the molecular layer of the hippocampus and the subiculum. Thus, the enkephalinlike immunoreactive nerve terminals are all located in areas which contain opioid binding sites. This suggests that the opioid peptide-opioid receptor systems may regulate hippocampal neuronal activity via neurotransmission or neuromodulation. However, a high or medium number of opioid binding sites occur over the pyramidal cell bodies and the dentate granule cell bodies, and these opioid binding sites are not in close contact with the major enkephalinergic systems. Such binding sites could represent newly synthesized opioid receptors ready for the enkephalinergic synapses of the cells and/or internalization of opioid receptors after stimulation at the synapses. Another possibility is the existence of cytoplasmic opioid binding sites (possibly t-RNA synthetase) with specific intracellular functions.