Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A₁ receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area.
2. The concentration-response curve for the inhibitory effects of the selective adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC₅₀=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC₅₀=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope.
3. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non-selective mGluR agonist (which activates both group I and II mGluR), (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; 100 μM). L-CCG-I had no consistent effects and ACPD (100 μM) decreased by 19.4±1.8% (n=4) the fe.p.s.p. slope.
4. The concentration-response curve for the inhibitory effects of CPA (2–100 nM) on the fe.p.s.p. slope (EC₅₀=8.2 (6.9–9.6) nM) was displaced to the right by the group III mGluR selective agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 25 μM) (EC₅₀=17.7 (13.1–21.9) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by L-AP4 (25 μM) was blocked in the presence of the mGluR antagonist (selective for the group III mGluR), (R,S)-α-methyl-4-phosphonophenylglycine (MPPG; 200 μM).
5. Both the direct effect of DHPG on synaptic transmission and the attenuation of the inhibitory effect of CPA (10 nM) were prevented in the presence of the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C.
6. The attenuation by L-AP4 (25 μM) of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope was also prevented by the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C. But this effect seemed to be distinct from the direct effect of L-AP4 (25 μM) on synaptic transmission, which was not modified by the protein kinase C selective inhibitors.
7. We conclude that agonists of metabotropic glutamate receptors (Groups I and III) are able to attenuate the inhibitory effects of adenosine A₁ receptor activation in the hippocampus. This interaction may have pathophysiological relevance in hypoxia, in which there is marked release of both excitatory amino acids and the important endogenous neuroprotective substance, adenosine.

     

大鼠代谢型谷氨酸受体第5亚型基因片段的克隆

从大鼠的尾壳核组织中提取总 RNA,以 RT-PCR方法扩增出大鼠 m Glu R5 长度约 43 5 bp的 c DNA片段。将这一片段克隆到 PGEM-T载体中进行序列分析 ,结果证实所克隆的 c DNA是编码正确的大鼠 m Glu R5 的一段基因序列。克隆的这段大鼠m Glu R5 特异性基因片段可用于制作探针 ,利用原位杂交技术检测其 m RNA在正常或异常状况下的表达 ;也可制作反意 c DNA或反意 m RNA以研究 m Glu R5 在生理或病理状态下的作用 ;还可进行反意 c DNA或反意 m RNA基因治疗。总而言之 ,克隆的这段基因 ,对研究 m Glu R5 在生理及病理条件下的功能变化 ,以及对与其有关疾病的基础研究和临床应用具有重要意义     

Of men and mice: Human X-linked retinoschisis and fidelity in mouse modeling

X-linked Retinoschisis (XLRS) is an early-onset transretinal dystrophy, often with a prominent macular component, that affects males and generally spares heterozygous females because of X-linked recessive inheritance. It results from loss-of-function RS1 gene mutations on the X-chromosome. XLRS causes bilateral reduced acuities from young age, and on clinical exam and by ocular coherence tomography (OCT) the neurosensory retina shows foveo-macular cystic schisis cavities in the outer plexiform (OPL) and inner nuclear layers (INL). XLRS manifests between infancy and school-age with variable phenotypic presentation and without reliable genotype-phenotype correlations. INL disorganization disrupts synaptic signal transmission from photoreceptors to ON-bipolar cells, and this reduces the electroretinogram (ERG) bipolar b-wave disproportionately to photoreceptor a-wave changes. RS1 gene expression is localized mainly to photoreceptors and INL bipolar neurons, and RS1 protein is thought to play a critical cell adhesion role during normal retinal development and later for maintenance of retinal structure. Several independent XLRS mouse models with mutant RS1 were created that recapitulate features of human XLRS disease, with OPL-INL schisis cavities, early onset and variable phenotype across mutant models, and reduced ERG b-wave to a-wave amplitude ratio. The faithful phenotype of the XLRS mouse has assisted in delineating the disease pathophysiology. Delivery to XLRS mouse retina of an AAV8-RS1 construct under control of the RS1 promoter restores the retinal structure and synaptic function (with increase of b-wave amplitude). It also ameliorates the schisis-induced inflammatory microglia phenotype toward a state of immune quiescence. The results imply that XLRS gene therapy could yield therapeutic benefit to preserve morphological and functional retina particularly when intervention is conducted at earlier ages before retinal degeneration becomes irreversible. A phase I/IIa single-center, open-label, three-dose-escalation clinical trial reported a suitable safety and tolerability profile of intravitreally administered AAV8-RS1 gene replacement therapy for XLRS participants. Dose-related ocular inflammation occurred after dosing, but this resolved with topical and oral corticosteroids. Systemic antibodies against AAV8 increased in dose-dependent fashion, but no antibodies were observed against the RS1 protein. Retinal cavities closed transiently in one participant. Technological innovations in methods of gene delivery and strategies to further reduce immune responses are expected to enhance the therapeutic efficacy of the vector and ultimate success of a gene therapy approach.     

Metabotropic glutamate receptor signaling is required for NMDA receptor-dependent ocular dominance plasticity and LTD in visual cortex

A feature of early postnatal neocortical development is a transient peak in signaling via metabotropic glutamate receptor 5 (mGluR5). In visual cortex, this change coincides with increased sensitivity of excitatory synapses to monocular deprivation (MD). However, loss of visual responsiveness after MD occurs via mechanisms revealed by the study of long-term depression (LTD) of synaptic transmission, which in layer 4 is induced by acute activation of NMDA receptors (NMDARs) rather than mGluR5. Here we report that chronic postnatal down-regulation of mGluR5 signaling produces coordinated impairments in both NMDAR-dependent LTD in vitro and ocular dominance plasticity in vivo. The data suggest that ongoing mGluR5 signaling during a critical period of postnatal development establishes the biochemical conditions that are permissive for activity-dependent sculpting of excitatory synapses via the mechanism of NMDAR-dependent LTD.Temporary monocular deprivation (MD) sets in motion synaptic changes in visual cortex that result in impaired vision through the deprived eye. The primary cause of visual impairment is depression of excitatory thalamocortical synaptic transmission in layer 4 of visual cortex (1–3). The study of long-term depression (LTD) of synapses, elicited in vitro by electrical or chemical stimulation, has revealed many of the mechanisms involved in deprived-eye depression (4). In slices of visual cortex, LTD in layer 4 is induced by NMDA receptor (NMDAR) activation and expressed by posttranslational modification and internalization of AMPA receptors (AMPARs) (5, 6). MD induces identical NMDAR-dependent changes in AMPARs, and synaptic depression induced by deprivation in vivo occludes LTD in visual cortex ex vivo (6–8). Manipulations of NMDARs and AMPAR trafficking that interfere with LTD also prevent the effects of MD (7, 9–11).Although NMDAR-dependent LTD is widely expressed in the brain (12, 13), it is now understood that different circuits use different mechanisms for long-term homosynaptic depression (14). For example, in the CA1 region of hippocampus, synaptic activation of either NMDARs or metabotropic glutamate receptor 5 (mGluR5) induces LTD. In both cases, depression is expressed postsynaptically as a reduction in AMPARs, but these forms of LTD are not mutually occluding and have distinct signaling requirements (15). A defining feature of mGluR5-dependent postsynaptic LTD in CA1 is a requirement for the immediate translation of synaptic mRNAs (16). In visual cortex, there is evidence that induction of LTD in layers 2–4 requires NMDAR activation, whereas induction of LTD in layer 6 requires activation of mGluR5 (17, 18).The hypothesis that mGluRs, in addition to NMDARs, play a key role in visual cortical plasticity can be traced back more than 25 y to observations that glutamate-stimulated phosphoinositide turnover, mediated in visual cortex by mGluR5 coupled to phospholipase C, is elevated during the postnatal period of heightened sensitivity to MD (19). Early attempts to test this hypothesis were inconclusive owing to the use of weak and nonselective orthosteric compounds (20–22); however, subsequent experiments did confirm that NMDAR-dependent LTD occurs normally in layers 2/3 of visual cortex in Grm5 knockout mice (23).The idea that mGluR5 is critically involved in visual cortical plasticity in vivo was rekindled with the finding that deprived-eye depression fails to occur in layer 4 of Grm5^+/− mutant mice (24). This finding was unexpected because, as reviewed above, a considerable body of evidence has implicated the mechanism of NMDAR-dependent LTD in deprived-eye depression. In the present study, we reexamined the role of mGluR5 in LTD and ocular dominance plasticity in layer 4, using the Grm5^+/− mouse and a highly specific negative allosteric modulator, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), that has proven suitable for chronic inhibition of mGluR5 (25, 26). Our data show that NMDAR-dependent LTD and deprived-eye depression in layer 4 require mGluR5 signaling during postnatal development.     

氧糖剥夺引起MF-CA3通路突触传递LTD现象及其机制研究

目的:观察氧糖剥夺引起的MF-CA3通路突触传递的长时程抑制(long-term depression, LTD)现象,并探讨其分子机制?方法:选择出生后2~3周的Sprague Dawley(SD)乳大鼠,制备海马脑片,通过刺激苔藓纤维,采用膜片钳的方法记录突触后电流?氧糖剥夺15 min后,观察突触传递的变化情况?分别采用犬尿喹啉酸阻断α-氨基羟甲基恶唑丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid,AMPA)受体?LY341495阻断代谢型谷氨酸受体﹑BAPTA螯合细胞内钙离子,观察对LTD的可能影响?结果:①氧糖剥夺15 min能引起MF-CA3通路AMPA受体介导的突触电流减弱,产生LTD现象;②若氧糖剥夺过程中运用阻断剂阻断AMPA受体的激活,洗脱后并不影响LTD的产生,而阻断代谢型谷氨酸受体可以使AMPA受体电流的LTD现象消失;③除去外液中的钙离子或用BAPTA螯合细胞内的钙离子均能使LTD现象消失?结论:氧糖剥夺引起MF-CA3通路上AMPA受体介导的突触电流持续减弱,呈现LTD现象?这种LTD现象依赖于代谢型谷氨酸受体的激活及胞内外钙离子浓度升高,但不依赖氧糖剥夺过程中AMPA受体的激活?     

Alcohol consumption increases basal extracellular glutamate in the nucleus accumbens core of Sprague–Dawley rats without increasing spontaneous glutamate release

Glutamate neurotransmission in the nucleus accumbens core (NAc) mediates ethanol consumption. Previous studies using non‐contingent and voluntary alcohol administration in inbred rodents have reported increased basal extracellular glutamate levels in the NAc. Here, we assessed basal glutamate levels in the NAc following intermittent alcohol consumption in male Sprague‐Dawley rats that had access to ethanol for 7 weeks on alternating days. We found increased basal NAc glutamate at 24 h withdrawal from ethanol and thus sought to identify the source of this glutamate. To do so, we employed a combination of microdialysis, slice electrophysiology and western blotting. Reverse dialysis of the voltage‐gated sodium channel blocker tetrodotoxin did not affect glutamate levels in either group. Electrophysiological recordings in slices made after 24 h withdrawal revealed a decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency relative to controls, with no change in sEPSC amplitude. No change in metabotropic glutamate receptor 2/3 (mGlu2/3) function was detected as bath application of the mGlu2/3 agonist LY379268 decreased spontaneous and miniature EPSC frequency in slices from both control and ethanol‐consuming rats. The increase in basal glutamate was not associated with changes in the surface expression of GLT‐1, however, a decrease in slope of the no‐net‐flux dialysis function was observed following ethanol consumption, indicating a potential decrease in glutamate reuptake. Taken together, these findings indicate that the increase in basal extracellular glutamate occurring after chronic ethanol consumption is not mediated by an increase in action potential‐dependent glutamate release or a failure of mGlu2/3 autoreceptors to regulate such release.     

Modulating mechanosensory afferent excitability by an atypical mGluR

Mechanotransduction by proprioceptive sensory organs is poorly understood. Evidence was recently shown that muscle spindle and hair follicle primary afferents (lanceolates) constantly release glutamate from synaptic‐like vesicles (SLVs) within the terminals. The secreted glutamate activates a highly unusual metabotropic glutamate receptor (mGluR) to modulate the firing rate (spindles) and SLV recycling (lanceolates). This receptor has yet to be isolated and sequenced. To further investigate this receptor's pharmacology, ligands selective for classical mGluRs have been recently characterised for their ability to alter stretch‐evoked spindle firing and SLV endocytosis in these different endings. Here, it is described how the results of these screens facilitated the development of novel compounds to be used in the process of isolating and sequencing of this non‐canonical mGluR. This study shows how the compounds were tested for their ability to alter stretch‐evoked afferent firing in muscle spindles and SLV endocytosis in the lanceolate endings of hair follicles to ensure they maintained their ability to bind to the receptor. For the development of novel compounds, kainate was chosen as the parent ligand due to its potency and ease of chemical modification. Novel kainate derivatives were then synthesised and tested to find potent analogues suitable for ‘click‐chemistry’, an established technique for relatively quick, cheap, stereospecific and high‐yield chemical modifications (Angewandte Chemie (International ed. in English), 40, 2001, pp2004). Of the novel kainate analogues developed, unfortunately ZCZ49 and ZCZ50 lost the ability to produce a significant change in spindle stretch‐evoked firing. However, ZCZ90 was as potent as kainate, increasing firing by a similar margin at 1 μm (n = 8; P < 0.001). The addition of either a biotin or a fluorescein side group to ZCZ90, using the click‐chemistry technique, did not affect the potency and hence these compounds will be used in further studies of the receptor. As well as the development of these compounds, the study found not only many similarities, but also some key differences between the two types of primary mechanosensory endings investigated. These differences must be taken into account in further study. However, they also present an intriguing opportunity for these receptors to be targeted selectively to modulate ending sensitivity as treatments for muscle spasm in multiple sclerosis and spinal cord injury, and possibly even baroreceptor firing to treat hypertension.     

Distinct trafficking and expression mechanisms underlie LTP and LTD of NMDA receptor‐mediated synaptic responses

Although an increasing number of studies have demonstrated the plasticity of NMDA receptor‐mediated synaptic transmission, little is known about the molecular mechanisms that underlie this neurologically important process. In a study of NMDAR‐mediated synaptic responses in hippocampal Schaffer‐CA1 synapses whose AMPA receptor (AMPAR) activity is totally blocked, we uncovered differences between the trafficking mechanisms that underlie the long‐term potentiation (LTP) and long‐term depression (LTD) that can be induced in these cells under these conditions. The LTP‐producing protocol failed to induce a change in the amplitude of NMDAR‐mediated postsynaptic currents (NMDAR EPSCs) in the first 5–10 min, but induced gradual enhancement of NMDAR EPSCs thereafter that soon reached a stable magnitude. This “slow” LTP of NMDAR EPSCs (LTP_NMDA) was blocked by inhibiting exocytosis or actin polymerization in postsynaptic cells. By contrast, LTD of NMDAR EPSCs (LTD_NMDA) was immediately inducible, and, although it was blocked by the actin stabilizer, it was unaffected by exocytosis or endocytosis inhibitors. Furthermore, concomitant changes in the decay time of NMDAR EPSCs suggested that differential switches in NR2 subunit composition accompanied LTP_NMDA and LTD_NMDA, and these changes were blocked by the calcium buffer BAPTA or an mGluR antagonist. Our results suggest that LTP_NMDA and LTD_NMDA utilize different NMDAR trafficking pathways and express different ratios of NMDAR subunits on the postsynaptic surface. © 2009 Wiley‐Liss, Inc.     

Elevated glycogen synthase kinase-3 activity in Fragile X mice: key metabolic regulator with evidence for treatment potential

Significant advances have been made in understanding the underlying defects of and developing potential treatments for Fragile X syndrome (FXS), the most common heritable mental retardation. It has been shown that neuronal metabotropic glutamate receptor 5 (mGluR5)-mediated signaling is affected in FX animal models, with consequent alterations in activity-dependent protein translation and synaptic spine functionality. We demonstrate here that a central metabolic regulatory enzyme, glycogen synthase kinase-3 (GSK3) is present in a form indicating elevated activity in several regions of the FX mouse brain. Furthermore, we show that selective GSK3 inhibitors, as well as lithium, are able to revert mutant phenotypes of the FX mouse. Lithium, in particular, remained effective with chronic administration, although its effects were reversible even when given from birth. The combination of an mGluR5 antagonist and GSK3 inhibitors was not additive. Instead, it was discovered that mGluR5 signaling and GSK3 activation in the FX mouse are coordinately elevated, with inhibition of mGluR5 leading to inhibition of GSK3. These findings raise the possibility that GSK3 is a fundamental and central component of FXS pathology, with a substantial treatment potential.