Protective effect of LY379268, a selective group II metabotropic glutamate receptor agonist, on dizocilpine-induced neuropathological changes in rat retrosplenial cortex

In the present study, we examined the effects of LY379268, the group II metabotropic glutamate receptor (mGluR) agonist, on the neuropathological changes in the rat retrosplenial cortex induced by noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine ((+)-MK-801). Administration of LY379268 (1, 3, 10 mg/kg, i.p.) reduced dizocilpine (0.5 mg/kg, i.p.)-induced neuropathological changes in the retrosplenial cortex, in a dose-dependent manner. Co-administration of LY379268 (10 mg/kg, i.p.) with group II mGluR antagonist LY341495 (5 mg/kg, i.p.) blocked the effects of LY379268. Furthermore, LY379268 (10 mg/kg, i.p.) significantly reduced the expression of heat shock protein HSP-70, a marker of reversible neuronal injury, in the rat retrosplenial cortex after administration of dizocilpine (0.5 mg/kg, i.p.). Moreover, pretreatment with LY379268 (10 mg/kg, i.p.) significantly suppressed the increase in extracellular acetylcholine (ACh) levels in the retrosplenial cortex induced by administration of dizocilpine (0.5 mg/kg, i.p.). These results suggest that LY379268 has a protective effect on the neurotoxicity in the rat retrosplenial cortex after administration of NMDA receptor antagonists such as dizocilpine.     

The group 2 metabotropic glutamate receptor agonist LY379268 rescues neuronal, neurochemical and motor abnormalities in R6/2 Huntington's disease mice

Excitotoxic injury to striatum by dysfunctional cortical input or aberrant glutamate uptake may contribute to Huntington's disease (HD) pathogenesis. Since corticostriatal terminals possess mGluR2/3 autoreceptors, whose activation dampens glutamate release, we tested the ability of the mGluR2/3 agonist LY379268 to improve the phenotype in R6/2 HD mice with 120-125 CAG repeats. Daily subcutaneous injection of a maximum tolerated dose (MTD) of LY379268 (20mg/kg) had no evident adverse effects in WT mice, and diverse benefits in R6/2 mice, both in a cohort of mice tested behaviorally until the end of R6/2 lifespan and in a cohort sacrificed at 10weeks of age for blinded histological analysis. MTD LY379268 yielded a significant 11% increase in R6/2 survival, an improvement on rotarod, normalization and/or improvement in locomotor parameters measured in open field (activity, speed, acceleration, endurance, and gait), a rescue of a 15-20% cortical and striatal neuron loss, normalization of SP striatal neuron neurochemistry, and to a lesser extent enkephalinergic striatal neuron neurochemistry. Deficits were greater in male than female R6/2 mice, and drug benefit tended to be greater in males. The improvements in SP striatal neurons, which facilitate movement, are consistent with the improved movement in LY379268-treated R6/2 mice. Our data indicate that mGluR2/3 agonists may be particularly useful for ameliorating the morphological, neurochemical and motor defects observed in HD.     

The metabotropic glutamate receptor 5 antagonist MPEP and the mGluR2 agonist LY379268 modify disease progression in a transgenic mouse model of Huntington's disease

Chronic glutamate mediated excitotoxicity has been suggested to contribute to the pathogenesis of Huntington's disease (HD). Both, inhibition of glutamate release through stimulation of presynaptic metabotropic glutamate receptor (mGluR) 2 and blockade of postsynaptic mGluR5 have been demonstrated to be neuroprotective against excitotoxicity. R6/2 HD transgenic mice which express an expanded CAG triplet repeat serve as a well-characterized mouse model for HD with progressing neurological abnormalities and limited survival. We treated R6/2 HD transgenic mice with either the mGluR2 agonist LY379268 (1.2 mg/kg) or with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (100 mg/kg) orally from a presymptomatic stage until death to investigate their potential disease modifying effects. We found that survival time in both the MPEP treated mice and the LY379268 treated mice was significantly increased in comparison to placebo treated transgenic controls (14.87+/-0.14 and 14.22+/-0.11 weeks versus 12.87+/-0.11 weeks, respectively). Additionally, the progressive decline in motor coordination of HD transgenic mice as tested with the rotarod test was significantly attenuated in MPEP- but not in LY379268-treated mice. Early pathological hyperactivity, which can be found in placebo treated HD transgenic mice, was significantly attenuated by both MPEP and LY379268 treatment. Immunohistologial examination of HD characteristic neuronal intranuclear inclusion (NII), however, demonstrated no effect on NII formation by either of the treatments applied. These data suggest that inhibition of glutamate neurotransmission via specific interaction with mGluRs might be interesting for both inhibition of disease progression as well as early symptomatic treatment in HD.     

代谢型谷氨酸受体1亚型选择性拮抗剂LY367385对抗大鼠缺血性脑水肿

目的研究代谢型谷氨酸受体1亚型（mGluR1）选择性拮抗剂LY367385对大鼠缺血性脑水肿的影响.方法Wistar雄性大鼠（280～320g）线栓法复制大脑中动脉闭塞（MCAO）脑缺血模型.动物随机分为生理盐水（NS）对照组、LY367385给药组及MK-801给药组,于MCAO后1min,侧脑室内注射NS或LY367385（500nmol）5μl,或腹腔注射MK-801（1mg/kg）.各组动物分别于MCAO 6、24h进行神经病学评分、脑含水量测定及脑梗死面积测定.结果LY367385明显改善大鼠脑缺血引起的神经症状,而MK-801在MCAO 6h增加神经病学评分.LY367385降低大鼠MCAO引起的脑含水量增加,MK-801无明显作用.LY367385及MK-801均降低脑梗死面积百分率,且LY367385作用优于MK-801.结论LY367385能对抗大鼠脑缺血性脑水肿,作用明显优于MK-801.     

Modulation of photic response by the metabotropic glutamate receptor agonist t-ACPD

Glutamate is the primary excitatory transmitter in the hypothalamus. It conveys photic information to the suprachiasmatic nucleus of the hypothalamus, thereby entraining the circadian clock to environmental light cycles. While ionotropic glutamate receptors have been implicated in the transduction of photic information in suprachiasmatic nucleus cells, there is evidence that metabotropic glutamate receptors play a significant modulatory role. We investigated the effects of the metabotropic glutamate agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) on light-evoked phase responses in Syrian hamsters at three phase points: circadian time 6, a time when light has no effect on the circadian timing system; circadian time 13.5, when light evokes the maximum phase delay; circadian time 19, the maximum phase advance. We found that ACPD significantly increased the light-evoked phase shift at circadian time 13.5, and had no effect at other phase points tested. These data support a role for metabotropic glutamate receptors in the circadian photic signal transduction system.     

Suppression of presynaptic calcium influx by metabotropic glutamate receptor agonists in neonatal rat hippocampus

Mechanisms of presynaptic inhibition by metabotropic glutamate receptor (mGluR) agonists were investigated in neonatal rat hippocampal CA1 region using the optical recording technique recently developed. Following selective loading of presynaptic terminals with a fluorescent Ca²⁺ indicator dye rhod-2 AM, changes in Ca²⁺ signals and the corresponding field excitatory postsynaptic potentials (EPSPs) induced by single electrical stimuli to the Schaffer collateral-commissural (SCC) pathway were recorded simultaneously. Application of a mGluR agonist, 1 S,3 R-1-aminocyclopentane-1,3-dicar?ylic acid (1 S,3 R-ACPD; 100 μM) or (±)-1-aminocyclopentane-trans-1,3-dicar?ylic acid (trans-ACPD; 100 μM), reversibly reduced both the field EPSP and the presynaptic Ca²⁺ transient, and the quantitative relationship between them was quite similar to that observed during application of Cd²⁺, a non-selective Ca²⁺ channel blocker, or in a Ca²⁺-free solution. Application of 4-aminopyridine (4-AP; 1 mM), a blocker of certain subtypes of voltage-dependent K⁺ channels, significantly inhibited the 1 S,3 R-ACPD effect. Application of DCG-IV, a novel mGluR2/mGluR3-selective agonist, suppressed field EPSPs only slightly even at a high dose (3 μM). These results suggest that activation of presynaptic mGluR different from mGluR2/mGluR3 suppresses the action potential-triggered Ca²⁺ influx, probably via 4-AP-sensitive mechanisms, and thereby reduces glutamate release in neonatal rat hippocampal CA1 region.     

A rat model of severe neonatal hypoxic-ischemic brain injury.

BACKGROUND AND PURPOSE: Perinatal hypoxic-ischemic brain injury is a common problem with severe neurological sequelae. In this report we describe in detail a simple model of hypoxia-ischemia in the neonatal rat that gives rise to severe neocortical infarction and to selective hippocampal neuronal necrosis. METHODS: Seven-day-old Simonsen Wistar rat pups underwent bilateral carotid artery ligation under methoxyflurane anesthesia and, after a 4 to 6-hour recovery, were exposed to 60 minutes of hypoxia (6.5% O2); they were perfusion-fixed 3 days later for histological study. Brain temperature was monitored throughout this treatment. RESULTS: We found that 64 +/- 3% of neocortex above the rhinal sulcus was infarcted; this infarction was evenly distributed through the cerebral hemispheres. In the hippocampus, neuronal necrosis was selective for the internal (hilar) layers of granule cells of the dentate gyrus, with relative sparing of CA1 pyramidal cells. In addition, brain temperature was tightly controlled throughout the experimental manipulations. CONCLUSIONS: The present model is easy and sensitive and provides an infarct of sufficient severity and homogeneity to make it well suited for pharmacological and biochemical studies directed toward therapeutic amelioration and mechanisms of hypoxic-ischemic brain damage, respectively. In addition, the pattern of damage in the hippocampus is quite different from that seen in the adult brain, which should be helpful in studying the ontogeny of selective vulnerability.     

Distribution of metabotropic glutamate receptor mGluR5 immunoreactivity in rat brain

The receptor mGluR5 is a metabotropic glutamate receptor with messenger RNA abundantly present throughout cortex, hippocampus, and caudate/putamen that is also coupled to phosphatidyl inositide hydrolysis and calcium mobilization. In this study, the distribution of mGluR5 was examined in rat brain by immunocytochemistry. The antibody utilized is highly specific and does not cross react with the most closely related other metabotropic glutamate receptor, as determined by Western blot analysis of nonneuronal cells transfected with metabotropic receptor coding sequences. The receptor mGluR5 is widely expressed with the highest density in olfactory bulb, caudate/putamen, lateral septum, cortex, and hippocampus, as confirmed with both immunocytochemistry and Western blot analysis. Electron microscopic studies in hippocampus and cortex indicate that the labeling is mostly on membranes of dendritic spines and shafts. Light and electron microscopic evidence indicates that some mGluR5 immunoreactivity is located in presynaptic axon terminals, suggesting that mGluR5 may function as a presynaptic receptor.     

Group II selective metabotropic glutamate receptor agonists and local cerebral glucose use in the rat.

The novel mGluR agonist LY354740 and a related analogue LY379268 are selective for mGluR2/3 receptors and are centrally active after systemic administration. In this study, rates of local cerebral glucose use were measured using the [14C]2-deoxyglucose autoradiographic technique to examine the functional consequences of their systemic administration in the conscious rat. Both LY354740 (0.3, 3.0, 30 mg/kg) and LY379268 (0.1, 1.0, 10 mg/kg) produced dose-dependent changes in glucose use. After LY354740 (3.0mg/kg), 4 of the 42 regions measured showed statistically significant changes from vehicle-treated controls: red nuclei (-16%), mammillary body (-25%), anterior thalamus (-29%), and the superficial layer of the superior colliculus (+50%). An additional 15 regions displayed significant reductions in function-related glucose use (P < .05) in animals treated with LY354740 (30 mg/ kg). LY379268 (0.1, 1.0, 10 mg/kg) produced changes in glucose metabolism in 20% of the brain regions analyzed. Significant increases (P < .05) in glucose use were evident in the following: the superficial layer of the superior colliculus (+81%), locus coeruleus (+57%), genu of the corpus callosum (+31%), cochlear nucleus (+26%), inferior colliculus (+20%), and the molecular layer of the hippocampus (+14%). Three regions displayed significant decreases: mammillary body (-34%), anteroventral thalamic nucleus (-28%), and the lateral habenular nucleus (-24%). These results show the important functional involvement of the limbic system together with the participation of components of different sensory systems in response to the activation of mGluR2 and mGluR3 with LY354740 and LY379268.     

alpha-Phenyl-n-tert-butyl-nitrone attenuates hypoxic-ischemic white matter injury in the neonatal rat brain

White matter of the neonatal brain is highly sensitive to hypoxic-ischemic insult. The susceptibility of premature oligodendrocytes (OLs) to free radicals (FRs) produced during hypoxia-ischemia (HI) has been proposed as one of the mechanisms involved. To test this hypothesis, and to further investigate if the FR scavenger alpha-phenyl-N-tert-butyl-nitrone (PBN) attenuates hypoxic-ischemic white matter damage (WMD), postnatal day 4 (P4) SD rats were subjected to bilateral common carotid artery ligation (BCAL), followed by 8% oxygen exposure for 20 min. Pathological changes were evaluated on P6 and P9, 2 and 5 days after the HI insult. HI caused severe WMD including rarefaction, necrosis and cavity formation in the corpus callosum, external and internal capsule areas. OL injury was evidenced by degeneration of O4 positive OLs on P6. Disrupted myelination was verified by decreased immunostaining of myelin basic protein (MBP) on P9. Axonal injury was demonstrated by increased amyloid precursor protein (APP) immunostaining on both P6 and P9. Two lipid peroxidation end products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), showed a one-fold elevation within 1-24 h following HI. 4-HNE immunostaining was found to specifically localize in the white matter area. Furthermore, pyknotic O4+ OLs were double-labeled with 4-HNE. These findings suggest that FRs are involved in the pathogenesis of neonatal WMD. PBN (100 mg/kg, i.p.) treatment alleviated the pathological changes of WMD following HI. It improved the survival of O4 positive OLs, attenuated hypomyelination and reduced axonal damage. PBN treatment also decreased the brain concentration of MDA/4-HNE and positive 4-HNE staining in the white matter area. These findings indicate that in the current WMD model, PBN protects both OLs and axons, the two main components in the white matter, from neonatal HI insult. FR scavenging appears to be the primary mechanism underlying its neuroprotective effect.     

Dopamine partial agonist actions of the glutamate receptor agonists LY 354,740 and LY 379,268

Because glutamate compounds alter the release of dopamine and prolactin, the present study examined whether group II metabotropic receptor agonists, LY 354,740 and LY 379,268, had any direct in vitro action on dopamine D2 receptors on rat striatal tissue, cloned D2Long receptors, and prolactin release from anterior pituitary cells. In competition versus the D2-specific ligand [(3)H]domperidone, LY 354,740 had a dissociation constant of 24 nM at D2(High) (the functional high-affinity state of dopamine D2 receptors), while the value for LY 379,268 was 21 nM. LY 354,740 also stimulated by 50% the incorporation of [(35)S]-GTP-gamma-S at a concentration of 120 nM, but its maximal stimulation was only 22% of the maximum elicited by dopamine. LY 379,268 stimulated by 50% the incorporation of [(35)S]-GTP-gamma-S at 280 nM, but its maximal stimulation was also only 22% of the maximum elicited by dopamine. However, both LY 354,740 and LY 379,268 potently inhibited the dopamine-induced incorporation of [(35)S]-GTP-gamma-S with inhibitory Ki values of 43 nM and 30 nM, respectively. The release of prolactin from rat isolated anterior pituitary cells in culture was 50% inhibited by 20 nM LY 379,268 and by 100 nM LY 354,740. These Ki values are similar to those known for the mGluR II receptor, suggesting that these compounds may have both glutamate and dopamine actions in vivo. The dopamine agonist and antagonist actions of these compounds indicate that these drugs have properties of a dopamine partial agonist, and may, therefore, have antipsychotic action.     

Erythropoietin exerts neuroprotective effect in neonatal rat model of hypoxic-ischemic brain injury

Hypoxic-ischemic encephalopathy seen in survivors of perinatal asphyxia is a frequently encountered and a major clinical problem for which there is currently no effective treatment. Hematopoietic neuroprotective agents, such as erythropoietin (EPO) may rescue neurons from cell death in this setting. EPO is a cytokine hormone that has neuroprotective effect in vitro and in vivo. In this study, we evaluated the effect of posthypoxic EPO administration in an animal model of neonatal hypoxic-ischemic injury. Our results show that a single intracerebroventricular injection of EPO immediately after hypoxic-ischemic insult in neonatal rat model of hypoxic-ischemia reduced the extent of hypoxic-ischemic brain damage. The mean infarct volume assessed 7 days after hypoxia was significantly smaller in EPO-treated group than in the control group. These findings suggest that EPO may provide benefit after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.     

HIF-1alpha inhibition ameliorates neonatal brain injury in a rat pup hypoxic-ischemic model

In this study, we tested if caspase-3 inhibition decreased ischemia-induced Aβ elevation by reducing β-secretase (BACE1) activity. Changes in caspase-3, Aβ and BACE1 levels were detected in rat striatum on different days after middle cerebral artery occlusion using immunostaining. We found that the positive labeled cells of activated caspase-3, Aβ, and BACE1 were significantly and time-dependently increased in the ipsilateral striatum. The results of Western blotting and RT-PCR showed that caspase-3 inhibitor Z-DEVD-FMK reduced BACE1 mRNA and protein levels, and inhibited its protease activity, thereby decreasing the amount of APP C99 and Aβ in ischemic brains. Moreover, Z-DEVD-FMK reduced BACE1 and GFAP double-labeled cells, but not GFAP protein levels or GFAP-labeled cells, in the ipsilateral striatum. Thus, we demonstrated that caspase-3 inhibition attenuated ischemia-induced Aβ formation by reducing BACE1 production and activity. This finding provides a therapeutic strategy for preventing Aβ accumulation and reducing the risk of neurodegeneration after stroke.     

Changes in iron histochemistry after hypoxic-ischemic brain injury in the neonatal rat

Iron can contribute to hypoxic-ischemic brain damage by catalyzing the formation of free radicals. The immature brain has high iron levels and limited antioxidant defenses. The objective of this study was to describe the early alterations in nonheme iron histochemistry following a hypoxic-ischemic (HI) insult to the brain of neonatal rats. We induced a HI insult to the right cerebral hemisphere in groups of 7-day-old rats. Rats were anesthetized, then their brains were perfused and fixed at 0, 1, 4, 8, 24 hr, and 1, 2, and 3 weeks of recovery. Forty-micron-thick frozen sections were stained for iron using the intensified Perls stain. Increased iron staining was first detected within the cytoplasm of cells with pyknotic nuclei at 4 hr of recovery. Staining increased rapidly over the first 24 hr in regions of ischemic injury. By 7 days recovery, reactive glia and cortical blood vessels also stained. Increased staining in gray matter persisted at 3 weeks of recovery, whereas white matter tracts had fewer iron-positive cells compared to normal. The early increase in iron staining could be caused by an accumulation of iron posthypoxicischemic injury or a change in iron from nonstainable heme iron to stainable nonheme iron. Regardless of the source, our results indicate that there is an increase in iron available to promote oxidant stress in the neonatal rat brain following hypoxia-ischemia.     

Minocycline worsens hypoxic-ischemic brain injury in a neonatal mouse model

Hypoxic-ischemic encephalopathy (HIE) is a leading cause of mortality and morbidity during the perinatal period, and currently no therapeutic drug is available. Minocycline, an antibiotic, has recently been shown to have neuroprotective effects distinct from its antimicrobial effect in several neurological disorders including ischemic brain injury. We examined the effect of minocycline on neonatal hypoxic-ischemic brain injury by using histologic scoring in both mouse and rat models. Mouse (C57Bl/6) and rat (SD) pups were exposed to a unilateral hypoxic-ischemic insult at 8 and 7 days of age, respectively. Minocycline hydrochloride was administered according to protocols that were reported to provide neuroprotection in adult or neonatal rats. Seven days after the insult, we examined brain injury in Nissl stained sections. Although minocycline ameliorated brain injury in the developing rat, it increased injury in the developing mouse. This detrimental effect in the mouse was consistent across different regions (cortex, striatum, and thalamus), with both single and multiple injection protocols and with both moderate and high-dose treatment (P < 0.05). The mechanism of the contrasting effects in mouse and rat is not clear and remains to be elucidated. Minocycline has been used as an antibiotic in the clinical setting for decades; therefore, it may be considered for use in infants with hypoxic-ischemic brain damage, based on prior reports of neuroprotection in the rat. However, it is important to examine this drug carefully before clinical use in human infants, taking our data in the mouse model into consideration.     

Widespread brain distribution of the Drosophila metabotropic glutamate receptor

Glutamate is the predominant excitatory neurotransmitter in the vertebrate brain, whereas acetylcholine has been considered to play the same role in insects. Recent studies have, however, questioned the latter view by showing a rather general distribution of glutamate transporters. Here, we describe the expression pattern of the receptor DmGlu-A (DmGluRA), the unique homolog of vertebrate metabotropic glutamate receptors. Metabotropic glutamate receptors play important roles in the regulation of glutamatergic neurotransmission. Using a specific antibody, we report DmGluRA expression in most neuropile areas in both larvae and adults, but not in the lobes of the mushroom bodies. These observations suggest a key role for glutamate in the insect brain.     

Additive neuroprotection by metabotropic glutamate receptor subtype-selective ligands in a rat Parkinson's model

Pharmacological activation of group III metabotropic glutamate receptors (mGluR) or inhibition of group I mGluR by subtype-selective ligands is neuroprotective in experimental models of Parkinson's disease. The aim of this study was to investigate whether targeting both receptor subtypes simultaneously produces enhanced neuroprotection. Rodents bearing a 6-hydroxydopamine lesion were intranigrally administered either the group III mGluR agonist L-(+)-2-amino-4-phosphonobutyric acid or the group I mGluR antagonist 2-methyl-6-(phenylethynyl)pyridine, alone or in combination. Coadministration of L-(+)-2-amino-4-phosphonobutyric acid and 2-methyl-6-(phenylethynyl)pyridine resulted in robust nigrostriatal neuroprotection that was significantly increased compared with either compound alone. These data suggest that targeting multiple mGluR subtypes with low doses of selective ligands may provide an enhanced therapeutic response in experimental models of Parkinson's disease.     

Immunocytochemical localization of metabotropic glutamate receptor type 1 alpha and tubulin in rat brain

The distribution of mGlu1 alpha receptor and tubulin was immunocytochemically examined in the rat cerebellar cortex and primary rat cortical neurons at both immunofluorescence and electron microscopic level. In cryosections from rat cerebellar cortex mGlu1 alpha receptor immunoreactivity was expressed in cell bodies and dendrites of Purkinje and basket cells of the cerebellar molecular layer. Tubulin immunoreactivity was concentrated in the dendritic tree of the cerebellar molecular layer, as well as in the granule cell layer. In primary rat cortical neurons, both proteins colocalized throughout the proximal and distal dendrites of these cells. At the electron microscopic level, the receptor was present in dendritic shafts and dendritic spines of Purkinje cells at perisynaptic sites of asymmetrical synapses. Immunoreactivity corresponding to tubulin was associated with the plasma membrane of dendritic shafts of Purkinje cells, as well as throughout its cytoplasm as part of the cytoskeletal components. Interestingly, double labeling for both proteins reveals an association of tubulin with mGlu1 alpha receptor at the plasma membrane level of dendritic shafts of Purkinje cells. This suggests that tubulin interacts with mGlu1 alpha receptor and may be involved in the anchoring of the receptor to the plasma membrane.     

A potent metabotropic glutamate receptor agonist: electrophysiological actions of a conformationally restricted glutamate analogue in the rat spinal cord and Xenopus oocytes

The (2S,3S,4S) isomer of alpha-(carboxycyclopropyl)glycine (L-CCG-I), a conformationally restricted glutamate analogue, caused a marked depolarization of motoneurons in the isolated rat spinal cord, which was almost insensitive to CPP and CNQX. Depolarizing responses to L-CCG-I were markedly decreased by reducing the temperature of the bathing fluid. Similar results were obtained in the case of trans-ACPD, which is a metabotropic glutamate receptor agonist, but the depolarizing action of L-CCG-I was more potent than that of trans-ACPD. In Xenopus oocytes injected with poly(A)+ mRNA extracted from the rat brain, L-CCG-I induced significant oscillatory chloride currents, suggesting that L-CCG-I is a potent agonist for metabotropic-type glutamate receptors.