Kelly I. Griffey

Induction or protection of limbic seizures in mice by mGluR subtype selective agonists

The behavioral consequences of metabotropic glutamate receptor (mGluR) activation were investigated following intracerebral administration of the mGluR selective agonists (RS)3,5-dihydroxyphenyl-glycine (3,5-DHPG), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD), (1R,3S)-1-aminocyclopentane-1,3-dicarboxylate (1R,3S-ACPD), L-2-amino-4-phosphonobutyrate (L-AP4), L-serine-O-phosphate (L-SOP) and (2S,3S,4S)alpha-(carboxycyclopropyl)glycine (L-CCGI) into the thalamus in mice. Injections of 3,5-DHPG, 1S,3R-ACPD and L-CCGI produced dose-dependent increases in limbic seizures with a potency order of 3,5-DHPG = 1S,3R-ACPD > L-CCGI. This effect of 1S,3R-ACPD was stereoselective, since the inactive isomer (1R,3S-ACPD) did not elicit seizure activity. Limbic seizures induced by the phosphoinositide-coupled mGluR subtype selective agonist 3,5-DHPG were attenuated by the mGluR antagonist L-2-amino-3-phosphonopropanoic acid (L-AP3) and dantrolene, inhibitors of mGluR-mediated intracellular calcium mobilization. Interestingly, L-AP4, L-SOP and low doses of L-CCGI also protected against 3,5-DHPG seizures. These data indicate that mGluR agonist-induced limbic seizures in mice are mediated by activation of phosphoinositide-coupled mGluRs. Furthermore, these seizures can be protected against by activation of mGluRs that are negatively-linked to cAMP formation.

The anxiolytic action of mGlu2/3 receptor agonist, LY354740, in the fear-potentiated startle model in rats is mechanistically distinct from diazepam.

The fear-potentiated startle paradigm has been characterized for drugs that act via ionotropic (NMDA and AMPA/kainate receptor) glutamate receptor mechanisms. Previous studies have shown that the potent systemically active mGlu2/3 receptor agonist, LY354740, effectively reduced the expression of fear-potentiated startle responses in rats. The present study examined the effects of LY354740 in a pre- versus post-fear conditioning paradigm and compared the effects to diazepam. Diazepam (0.3, 0.6, and 1.0 mg/kg ip) attenuated both pre- and post-fear conditioning startle responses in a dose-related manner. In contrast, LY354740 (0.03, 0.3, and 3.0 mg/kg ip) did not disrupt preconditioning startle responses at doses that attenuated post-fear conditioning responses. The benzodiazepine antagonist, flumazenil, at a dose (2 mg/kg sc) that did not alter fear-potentiated startle per se, selectively reversed suppression of fear responses to diazepam (0.6 mg/kg ip) while not affecting fear suppression induced by LY354740 (0.3 mg/kg ip). At a dose of 1 mg/kg ip, the mGlu2/3 receptor antagonist, LY341495, did not disrupt fear-enhanced startle per se, but completely reversed the postconditioning anxiolytic effects of LY354740 in this model. This dose of LY341495 had no effect on fear suppression by diazepam. These results demonstrate that fear suppression by diazepam and LY354740 involves different neuronal mechanisms. While diazepam acts via the facilitation of GABAergic transmission, LY354740 induces its actions via the glutamatergic system, specifically mGlu2/3 receptor activation. Furthermore, in contrast to disruption of fear conditioning as well as fear suppression by diazepam, LY354740 had selective effects on fear expression, suggesting anxiolytic actions without the associated memory impairment.

Intracerebral 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) produces limbic seizures that are not blocked by ionotropic glutamate receptor antagonists

The functional role of metabotropic glutamate receptor (mGluR) activation was investigated following intracerebral administration of 1S,3R-ACPD in mice. Injections of 1S,3R-ACPD (50-800 nmol in 5 microliters) into the thalamus produced a dose-dependent increase in limbic seizures. These effects were stereoselective since 1R,3S-ACPD, did not elicit seizure activity. Pharmacologically, limbic seizures were attenuated by the mGluR partial agonist/antagonist L-2-amino-3-phosphonopropionate (L-AP3) and dantrolene, an inhibitor of intracellular calcium mobilization, but not by D-AP3 or ionotropic glutamate receptor antagonists (MK-801 or GYKI-52466). Thus, activation of mGluRs by 1S,3R-ACPD in mice, induces limbic seizures that may involve the mobilization of intracellular calcium stores.

Inhibition of group I metabotropic glutamate receptor responses in vivo in rats by a new generation of carboxyphenylglycine-like amino acid antagonists

A series of novel group I metabotropic glutamate receptor (mGlu) antagonists have been designed on the basis of the 4-carboxyphenylglycine pharmacophore. The compounds are either mGlu1 receptor selective or equipotent for both mGlu1 and mGlu5 receptors and have IC(50) values ranging from 1 to 30 microM determined by phosphoinositide hydrolysis (PI) assay in vitro. All the compounds produced dose-dependent inhibition of group I mGlu receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced limbic seizure responses in mice with ED(50) values ranging from 9 nmol for LY393053 to 138 nmol for LY339840 after intracerebroventricular injection and were more potent than the mGlu1 receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (ED(50)=477 nmol). Further antagonist actions were also demonstrated in a model of (RS)-DHPG-induced PI hydrolysis in vivo such that LY367385 and the active cis isomer of LY393053 produced dose-dependent inhibition of PI responses in both cerebellum and hippocampus. Cis LY393053 also inhibited hippocampal PI responses when administered intraperitoneally at a dose of 30 mg/kg. These compounds define a new series of group I mGlu receptor antagonists which may serve as useful experimental tools.

The Selective Estrogen Receptor Modulator, Raloxifene: A Segment II/III Delivery Study in Rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company as a therapeutic agent for postmenopausal osteoporosis. Raloxifene was administered orally by gavage at doses of 0, 0.1, 1, or 10 mg/kg/d to female CD rats (25/group) on Gestation Day 6 (GD 6) through Postpartum Day 20 (PD 20). Females were allowed to deliver and maintain their progeny until PD 21. All dead pups and pups culled on PD 1 were given internal and external examinations. One pup/sex/litter was assigned to each of the following assessment groups: 1) the primary pair for the F1 generation study, in which survival, growth, development, behavior, indicators of sexual maturation, and reproductive performance were evaluated; 2) terminal necropsy evaluations at PD 21; 3) terminal necropsy evaluations at 60 d of age; and 4) assessments of immune function at 5 to 6 weeks of age. At termination on PD 21, 60, or approximately 140, a necropsy was performed; crown rump and tibia lengths were measured; pituitary weights were taken; and a portion of the anterior pituitary was retained for growth hormone, luteinizing hormone, and prolactin content determinations (control and 10-mg/kg groups only). The remainder of the pituitary and reproductive tissues were retained for histologic evaluations. Dose-related depressions in maternal body weight and food consumption occurred during gestation. Mean gestation length was increased at 1 and 10 mg/kg. Delayed, extended, and/or disrupted parturition occurred in dams given 10 mg/kg, which resulted in a high incidence of maternal morbidity and/or death, increased numbers of dead pups, and the survival of only 66% of live pups to PD 21. Progeny body weights were not decreased at birth, but were depressed progressively in a dose-related manner during the 3-week lactation period. Negative geotaxis and incisor eruption were apparently accelerated in the 1- and 10-mg/kg groups, but eye opening was delayed at 10 mg/kg. Postweaning activity levels, auditory startle, and passive avoidance performance were not affected in the raloxifene groups. Dose-related decreases in spleen cellularity and thymus weights occurred in both sexes, but immune system function, as measured by splenic natural killer cell activity and antibody response to sheep red blood cells, was not affected. Postweaning body weights and growth parameters, as well as pituitary hormone content, were affected in both an age- and sex-specific manner. Preputial separation was not affected, but vaginal patency occurred ca 2 d earlier than controls in females from the 10-mg/kg group. Estrous cycles of the F1 females were not affected during the first two weeks after vaginal opening, but were disrupted at 12 to 14 weeks of age in the 10-mg/kg group. These females showed poorer mating and fertility indices, and litter size was reduced in the two females that were pregnant. Histologically, reproductive organs were not affected in males at any age or in females at PD 21. At PD 60, vaginal mucification occurred in females from the 0.1- and 1-mg/kg groups. At PD 140, the only finding was a high rate of uterine hypoplasia in the 10-mg/kg group, and this finding occurred in the absence of any concomitant ovarian or vaginal changes. These reproductive and developmental findings are consistent with estrogen antagonist activity of raloxifene.

Neonatal exposure to d,l-2-amino-3-phosphonopropionate (d,l-AP3) produces lesions in the eye and optic nerve of adult rats

Metabotropic glutamate receptors are a recently described receptor class with emerging importance in synaptic plasticity and brain development. Activation of metabotropic glutamate receptors results in several cellular secondary messenger events that are especially important during postnatal development. This study characterized the effects of D,L-2-amino-3-phosphonopropionate (D,L-AP3), an aspartic acid analog with agonist and antagonist activity at the metabotropic receptor, on the postnatal development of the rat eye and optic nerve. Sprague-Dawley rat pups were treated daily (i.p.) with saline or 500 mg/kg D,L-AP3 on postnatal days (PND) 4-10 or 10-14. After making clinical and ophthalmoscopic examinations, rats were necropsied between 65 and 70 days of age and light microscopic evaluations were made of eyes and optic nerves. Between postnatal days 10-20, all treated rats exhibited motor tremors, circling, and head tilt. Ophthalmoscopic lesions were more severe in rats treated on days 4-10 than days 10-14 and included decreased retinal vasculature, cataracts, and retinal dysplasia, hypoplasia, and detachment. All rats treated on days 4-10 had severe optic nerve atrophy/hypoplasia grossly and severe retinal atrophy, retinal detachment, and cataracts histologically. Seven of eight rats treated on days 10-14 had qualitatively similar but less severe lesions. Overall, rats treated with D,L-AP3 on PND 4-10 had earlier and more severe retinal and optic nerve lesions when compared to rats treated on PND 10-14. These data characterize the morphologic effects in adult rats exposed to D,L-AP3 as neonates and suggest a possible role for the metabotropic receptor in the postnatal development of retina and optic nerve.

The selective estrogen receptor modulator, raloxifene : Reproductive assessments following preimplantation exposure in mated female rats

Raloxifene is a nonsteroidal, selective estrogen receptor modulator being developed for postmenopausal osteoporosis. As part of an integrated reproductive toxicity assessment, two studies were conducted in which raloxifene was administered orally to CD rats during Gestation Days (GD) 0 through 5. In each study, animals received daily raloxifene doses of 0, 0.1, 1, or 10 mg/kg. In Study 1, GD 20 evaluations of maternal reproductive parameters identified dose-related increases in pre- and postimplantation loss, reductions in the numbers of corpora lutea and live conceptuses, and reduced fetal weight. The low fetal weights were consistent with an extent of morphologic development that corresponded to developmental ages up to 8 d younger than GD 20. Study 2 characterized the potential impact of this disrupted and apparently delayed implantation on gestation length, parturition, and progeny viability. Dams were allowed to deliver and rear their offspring through Postpartum Day 21. Gestation lengths were extended up to 1 week, and litter sizes were reduced in a dose-dependent manner. Nevertheless, parturition occurred normally and pup morphology, survival, and physical and behavioral development were unaffected.