Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.     

Modulation of breathing by mu1 and mu2 opioid receptor stimulation in neonatal and adult rats

Opioid modulation of breathing during postnatal development through to the adult was investigated in the rat. Respiratory frequency, tidal volume and minute volume were recorded in unanesthetized, unrestrained rat pups and adults using barometric plethysmography. Subjects were administered the highly selective mu opioid agonists dermorphin and fentanyl. Fentanyl, which readily crosses the blood-brain barrier, was included to ensure that developmental changes in blood-brain barrier restrictions did not mask some of the dermorphin effects in older neonates. Drugs were administered subcutaneously in neonates and adults, although dermorphin was given by intracerebroventricular route only in adults. In neonates, mu agonist administration caused a gasping-like pattern of breathing, characterized by a marked fall in frequency and a smaller increase in tidal volume. The gasping response was prevented by pre-treatment with the long-acting mu1 antagonist naloxonazine (NALZ). In the presence of NALZ, mu agonists elicited only a small, but significant, reduction in tidal volume. Both dermorphin and fentanyl showed more potent activity in younger pups than in older pups, possibly in the case of dermorphin because of developmental maturation of blood-brain barrier function. In adults, fentanyl and dermorphin both caused a reduction in frequency and minute volume. The response of adults to fentanyl, but not dermorphin, was prevented by NALZ. These results suggest that both mu1 and mu2 receptors contribute to opioid-induced respiratory depression during neonatal and adult life.     

Selective changes in mu opioid receptor properties induced by chronic morphine exposure.

Chronic infusion of morphine to guinea pigs produced selective changes in mu agonist binding properties in cerebrocortical membrane preparations. Employing the mu-selective opioid agonist [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) in direct binding studies and in competition of labeled antagonist binding, we found that the major changes were a decrease in the number of sites with high affinity for agonist, a small reduction in total receptor number, and a loss in the ability of guanosine 5'-[gamma-thio]triphosphate to regulate binding. A fraction of high-affinity mu receptors appeared to retain their high affinity for agonist and their sensitivity to guanine nucleotide analogue after the induction of morphine tolerance, possibly because the morphine concentrations achieved in brain were insufficient to uncouple all mu receptors from associated guanine nucleotide-binding regulatory proteins. Some membrane preparations were treated with pertussis toxin, which has been shown to functionally uncouple mu opioid receptors from their effector systems. In these preparations, a single agonist-affinity state of the receptor was observed. The apparent dissociation constant for this affinity state in pertussis toxin-treated membranes was similar to the lower-affinity state observed in preparations from morphine-tolerant animals. In contrast to the changes observed at mu opioid binding sites, no significant changes in agonist affinity or binding density were observed for selective delta or kappa agonists, consistent with the development of selective tolerance at mu receptors.     

mu opiate receptor: cDNA cloning and expression.

mu opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides.     

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.     

Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects

Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.     

大麻素受体在海马CA1区锥体神经元的功能表达

目的观察大麻素受体在孤立的海马CA1区锥体神经元的功能表达。方法将出生15~20d的Wistar大鼠取脑,急性分离出单个CA1区锥体神经元,用膜片钳技术记录神经元电活动,观察非选择性大麻素受体激动剂Win55212-2(5μmol/L)对神经元静息电位、动作电位、自发发放频率的影响。根据Win55212-2对膜电位的影响分为超极化组(n=7)和去极化组(n=6)。组织切片活性用MTT染色法检测。结果与给药前比较,超极化组神经元给药中动作电位频率和膜电压显著降低[0Hz vs(4.3±3.2)Hz,P0.05;(-57.0±4.6)mVvs(-54.1±3.8)mV,P0.01];与给药中比较,给药后动作电位频率及膜电压显著升高,差异有统计学意义(P0.01)。与给药前比较,去极化组神经元给药中动作电位频率显著降低,膜电压显著升高(P0.01);与给药中比较,给药后动作电位频率显著升高,膜电压显著降低,差异有统计学意义(P0.05)。结论 CA1区锥体神经元可能存在大麻素受体功能表达且不限于大麻素受体1;激活大麻素受体可能通过不同的机制起到抑制CA1区锥体神经元的作用。     

11beta-hydroxylase and aldosterone synthase expression in fetal rat hippocampal neurons

The central nervous system produces many of the enzymes responsible for corticosteroid synthesis. A model system to study the regulation of this local system would be valuable. Previously, we have shown that primary cultures of hippocampal neurons isolated from the fetal rat can perform the biochemical reactions associated with the enzymes 11beta-hydroxylase and aldosterone synthase. Here, we demonstrate directly that these enzymes are present within primary cultures of fetal rat hippocampal neurons.     

A glutamate receptor regulates Ca2+ mobilization in hippocampal neurons.

We investigated the effect of various excitatory amino acids on intracellular free Ca2+ concentration ( [Ca2+]i) in single mouse hippocampal neurons in vitro by using the Ca2+-sensitive dye fura-2. In normal physiological solution, glutamate, kainate, N-methyl-D-aspartate, and quisqualate all produced increases in [Ca2+]i. When all extracellular Ca2+ was removed, kainate and N-methyl-D-aspartate were completely ineffective, but quisqualate and glutamate were able to produce a spike-like Ca2+ transient, presumably reflecting the release of Ca2+ from intracellular stores. Ca2+ transients of similar shape could also be produced by the alpha 1-adrenergic agonist phenylephrine. After the production of a Ca2+ transient a second addition of quisqualate was ineffective unless intracellular stores were refilled by loading the cell with Ca2+ following depolarization in Ca2+-containing medium. None of the conventional excitatory amino acid receptor antagonists inhibited the Ca2+-mobilizing effects of quisqualate. Furthermore alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unable to produce Ca2+ mobilization in Ca2+-free medium, although it could produce Ca2+ influx in Ca2+-containing medium. Thus, glutamate can produce mobilization of Ca2+ from intracellular stores in hippocampal neurons by acting on a quisqualate-sensitive but AMPA-insensitive receptor. This receptor is therefore distinct from the quisqualate receptor that produces cell depolarization. The possibility that this Ca2+-mobilizing effect is mediated by inositol triphosphate production is discussed.     

AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na⁺/H⁺-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.     

Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception

To develop a genetic approach for the treatment of pain, we introduced a recombinant adeno-associated viral (rAAV) vector containing the cDNA for the mu-opioid receptor (muOR) into primary afferent neurons in dorsal root ganglia (DRGs) of rats, which resulted in a long-lasting (>6 months) increase in muOR expression in DRG neurons. The increase greatly potentiated the antinociceptive effects of morphine in rAAV-muOR-infected rats with and without inflammation. Perforated patch recordings indicated that the efficacy and potency of opioid inhibition of voltage-dependent Ca(2+) channels were enhanced in infected neurons, which may underlie the increase in opiate efficacy. These data suggest that transfer of opioid receptor genes into DRG cells with rAAV vectors may offer a new therapeutic strategy for pain management.     

Reduced hippocampal MT2 melatonin receptor expression in Alzheimer's disease

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.     

Effect of fluoride on expression of phosphorylated NMDAR protein in rat hippocampal neurons

<正>Objective To observe the expression of phosphorylated N-methyl-D-aspartate receptor (P-NMDAR) protein in hippocampal neurons of rats with fluorosis and explore the mechanism of neuronal damage caused by fluorosis.Methods Sixteen Sprague-Dawley rats within 24 h after     

Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain.

In membranes from SH-SY5Y human neuroblastoma cells differentiated with retinoic acid, the mu-selective agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) inhibited cAMP formation with an IC50 of 26 nM. Two separate antibodies raised against distinct regions of the Go alpha sequence attenuated the effect of DAMGO by 50-60%, whereas antibodies to Gi alpha 1,2 or Gi alpha 3 reduced the mu-opioid signal insignificantly or to a lesser extent. In contrast, inhibition of adenylyl cyclase by the delta-opioid agonist Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE; Pen = penicillamine) was very sensitive to the Gi alpha 1,2 antibody. In membranes from rat brain striatum, coupling of the mu opioid receptor to adenylyl cyclase was also maximally blocked by antibodies to Go alpha. After long-term treatment of the cells with DAMGO, the content of Go alpha was reduced by 26%, whereas the levels of Gi alpha 1,2, Gi alpha 3, and Gs alpha were unaltered. Addition of Go, purified from bovine brain, to membranes from pertussis toxin-treated SH-SY5Y cells restored the inhibition of adenylyl cyclase by DAMGO to 70% of that in toxin-untreated cells. To comparably restore the effect of DPDPE, much higher concentrations of Go were required. By demonstrating mediation of cAMP-dependent signal transduction by Go, these results describe (i) an additional role for this G protein present at a high concentration in brain, (ii) preferential, although not exclusive, interaction of mu and delta opioid receptors with different G protein subtypes in coupling to adenylyl cyclase, and (iii) reduced levels of Go following chronic opioid treatment of SH-SY5Y cells with mu opioids.     

Network synchronization in hippocampal neurons

Oscillatory activity is widespread in dynamic neuronal networks. The main paradigm for the origin of periodicity consists of specialized pacemaking elements that synchronize and drive the rest of the network; however, other models exist. Here, we studied the spontaneous emergence of synchronized periodic bursting in a network of cultured dissociated neurons from rat hippocampus and cortex. Surprisingly, about 60% of all active neurons were self-sustained oscillators when disconnected, each with its own natural frequency. The individual neuron’s tendency to oscillate and the corresponding oscillation frequency are controlled by its excitability. The single neuron intrinsic oscillations were blocked by riluzole, and are thus dependent on persistent sodium leak currents. Upon a gradual retrieval of connectivity, the synchrony evolves: Loose synchrony appears already at weak connectivity, with the oscillators converging to one common oscillation frequency, yet shifted in phase across the population. Further strengthening of the connectivity causes a reduction in the mean phase shifts until zero-lag is achieved, manifested by synchronous periodic network bursts. Interestingly, the frequency of network bursting matches the average of the intrinsic frequencies. Overall, the network behaves like other universal systems, where order emerges spontaneously by entrainment of independent rhythmic units. Although simplified with respect to circuitry in the brain, our results attribute a basic functional role for intrinsic single neuron excitability mechanisms in driving the network’s activity and dynamics, contributing to our understanding of developing neural circuits.Periodicity emerges as a key physiological characteristic at all levels of neuronal activity, from the dynamics of neurons at subthreshold potentials (1, 2), through rhythmic neuronal ensembles within local networks, and all of the way up to global oscillations measured by electroencephalography (EEG) (3). The range of observed frequencies is surprisingly wide, from the millisecond range typical for interspike intervals all of the way to several seconds in the case of slow EEG. Over the years, accumulating evidence and theory have attributed different mechanisms for the origin of each measured periodic activity. Within local networks, the role of known collective mechanisms for periodicity, such as the balance between excitatory and inhibitory neurons and recurrent network architecture (4–6), is often contrasted with single neuron contributions, for example, the role of pacemaker neurons in oscillatory network dynamics (7). Although the physiological properties of single neurons are diverse and well documented (2, 8), their role in emergent network oscillations was predicted theoretically (9), but has not been observed experimentally. This contribution may involve a subtle interplay between intrinsic excitability and network connectivity (10, 11).The connectivity and excitability together determine the behavior of the network. The connectivity can be decreased either at the receiving, postsynaptic neuron or at the sending, presynaptic neuron. The postsynaptic end is blocked by the use of appropriate synaptic blockers. The presynaptic end can be modified by decreasing [Ca²⁺]_o in the environment and effectively stopping synaptic vesicle release.To understand excitability, one must take into account the origin of membrane potential fluctuations, which can drive a single neuron to fire action potentials (APs). Although APs of coupled neurons are usually triggered via an “integrate and fire” process, there is also evidence for synapse-independent intrinsic processes capable of driving APs (11). Both synaptic and intrinsic processes affect the excitability state of the cell, which, in turn, determines how close to threshold the cell hovers before firing.The excitability can also be modulated by the balance between intracellular and extracellular ionic concentrations. Changes in the extracellular ionic environment can be globally controlled and take effect immediately without relying on adaptive mechanisms. The effect of changes in the ionic environment on intrinsic excitability can be striking (7, 12). The potassium concentration directly affects the resting membrane potential of the neuron, but other subtle effects can also arise from divalent ions that affect specific channel activity, and thus the internal excitability of the cell.In particular, calcium plays an important role in controlling collective behavior by participating in synaptic transmission between neurons, regulating the release of neurotransmitter vesicles (13). Furthermore, calcium is important for determining the single cell excitability, for example, by regulating leak currents into the neuron via the NALCN (sodium leak channel) (14, 15).In this study, we modified both excitability and connectivity by systematically varying the network environment, and we used multielectrode arrays (MEAs) to follow the spontaneous activity of up to 59 single neurons continuously and simultaneously in a dissociated culture. The large-scale network bursting that is characteristic of dissociated culture activity is easily monitored with single spike resolution in this system (16). The precise control of external conditions, along with a high temporal resolution and the relatively large number of recorded neurons in this system, makes it possible to search for the role of heterogeneous intrinsic properties of single neurons in the emergence of synchronized network bursts.     

Kindling produces long-lasting and selective changes in gene expression of hippocampal neurons.

To test the hypothesis that repeated subconvulsive stimulations required to induce kindling can permanently alter gene expression of hippocampal neurons, we used Northern and in situ hybridization analyses to measure steady-state mRNA levels encoding several phenotypic proteins. mRNA encoding a membrane-bound protein, ligatin, was significantly reduced in kindled brains relative to naive and sham control animals. This change in gene expression persisted for over 4 months after kindling, was associated with a decrease in ligatin protein expression, was not produced by single or multiple seizures that did not induce the kindling phenomena, and was blocked by MK801. These results provide direct evidence that kindling can cause persistent changes in the expression of specific genes in hippocampal neurons and suggest that N-methyl-D-aspartate receptor-activated changes in gene expression may be a basic molecular mechanism underlying some of the long-lasting plasticity changes seen in kindling or models of long-term memory.