The association of polygenic risk for schizophrenia, bipolar disorder, and depression with neural connectivity in adolescents and young adults: examining developmental and sex differences.

Neurodevelopmental abnormalities in neural connectivity have been long implicated in the etiology of schizophrenia (SCZ); however, it remains unclear whether these neural connectivity patterns are associated with genetic risk for SCZ in unaffected individuals (i.e., an absence of clinical features of SCZ or a family history of SCZ). We examine whether polygenic risk scores (PRS) for SCZ are associated with functional neural connectivity in adolescents and young adults without SCZ, whether this association is moderated by sex and age, and if similar associations are observed for genetically related neuropsychiatric PRS. One-thousand four-hundred twenty-six offspring from 913 families, unaffected with SCZ, were drawn from the Collaborative Study of the Genetics of Alcoholism (COGA) prospective cohort (median age at first interview = 15.6 (12-26), 51.6% female, 98.1% European American, 41% with a family history of alcohol dependence). Participants were followed longitudinally with resting-state EEG connectivity (i.e., coherence) assessed every two years. Higher SCZ PRS were associated with elevated theta (3-7 Hz) and alpha (7-12 Hz) EEG coherence. Associations differed by sex and age; the most robust associations were observed between PRS and parietal-occipital, central-parietal, and frontal-parietal alpha coherence among males between ages 15-19 (B: 0.15-0.21, p < 10-4). Significant associations among EEG coherence and Bipolar and Depression PRS were observed, but differed from SCZ PRS in terms of sex, age, and topography. Findings reveal that polygenic risk for SCZ is robustly associated with increased functional neural connectivity among young adults without a SCZ diagnosis. Striking differences were observed between men and women throughout development, mapping onto key periods of risk for the onset of psychotic illness and underlining the critical importance of examining sex differences in associations with neuropsychiatric PRS across development.

Water intake and urinary hydration biomarkers in children.

BACKGROUND/OBJECTIVES: The aims of the study were as follows: (1) examine fluid intake and urinary hydration markers of children in Greece, (2) determine the calculated relative risk of hypohydration in children who did not meet the recommendations for daily water intake provided by the Institute of Medicine and the European Food Safety Authority compared with those who did and (3) analyze the efficacy of the recommendations as a method to achieve euhydration in children. SUBJECTS/METHODS: One hundred and fifty Greek boys and girls (age 9-13) recorded their fluid intake for 2 consecutive days. A 24-h urine collection was obtained during the second day. Fluid intake records were analyzed for total water intake from fluids (TWI-F), and urine samples were analyzed for osmolality, color, specific gravity and volume. Urine osmolality ⩾800 mmol/kg H2O was defined as hypohydration. RESULTS: Water intake from fluids was 1729 (1555-1905) and 1550 (1406-1686) ml/d for boys and girls, respectively. Prevalence of hypohydration was 33% (44% of boys, 23% of girls). Children who failed to meet TWI-F recommendations demonstrated a risk of hypohydration that was 1.99-2.12 times higher than those who met recommendations (P⩽0.01). Boys between 9 and 13 years displayed urine osmolality of 777 (725-830) mmol/kg, and urine specific gravity of 1.021 (1.019-1.022), which was higher than those in girls between 9-13 years (P⩽0.015), and >27% were classified as hypohydrated despite meeting water intake recommendations. CONCLUSIONS: Failure to meet TWI-F guidelines increased calculated relative risk of hypohydration in children. Boys between 9 and 13 years are at greater hazard regardless of meeting guidelines and may require greater water intake to avoid elevated urine concentration and ensure adequate hydration.

Assessment of hydration biomarkers including salivary osmolality during passive and active dehydration.

BACKGROUND/OBJECTIVES: Hydration state can be assessed via body mass change (BMΔ), serum and urine osmolality (Sosm, Uosm), urine-specific gravity (Usg) and urine volume (Uvol). As no hydration index has been shown to be valid in all circumstances, value exists in exploring novel biomarkers such as salivary osmolality (Vosm). Utilizing acute BMΔ as the reference standard, this research examined the efficacy of Sosm, Vosm, Uosm, Uvol and Usg, during passive (PAS) and active (ACT) heat exposure. SUBJECTS/METHODS: Twenty-three healthy men (age, 22±3 years; mass, 77.3±12.8 kg; height, 179.9±8.8cm; body fat, 10.6±4.5%) completed two randomized 5-h dehydration trials (36±1 °C). During PAS, subjects sat quietly, and during ACT, participants cycled at 68±6% maximal heart rate. Investigators measured all biomarkers at each 1% BMΔ. RESULTS: Average mass loss during PAS was 1.4±0.3%, and 4.1±0.7% during ACT. Significant between-treatment differences at -1% BMΔ were observed for Sosm (PAS, 296±4; ACT, 301±4 mOsm/kg) and Uosm (PAS, 895±207; ACT, 661±192 mOsm/kg). During PAS, only Uosm, Uvol and Usg increased significantly (-1 and -2% BMΔ versus baseline). During ACT, Vosm most effectively diagnosed dehydration 2% (sensitivity=86%; specificity=91%), followed by Sosm (sensitivity=83%; specificity=83%). Reference change values were validated for Sosm, Usg and BMΔ. CONCLUSIONS: The efficacy of indices to detect dehydration 2% differed across treatments. At rest (PAS), only urinary indices increased in concert with body water loss. During exercise (ACT), Sosm and Vosm exhibited the highest sensitivity and specificity. Sosm, Usg and BMΔ exhibited validity in serial measurements. These findings indicate hydration biomarkers should be selected by considering daily activities.

Evaluation of Uosm:Posm ratio as a hydration biomarker in free-living, healthy young women.

BACKGROUND/OBJECTIVES: Urinary and plasma indices are utilized to assess whole-body water balance in healthy adults, whereas the urine-to-plasma osmolality ratio (Uosm:Posm) rarely is. To explore the efficacy of Uosm:Posm as a hydration biomarker, diet records of 120 college women were analyzed (beverage water+food water=total fluid intake (TFI); 5 days) to identify habitual high-volume (HIGH) and low-volume (LOW) drinkers. SUBJECTS/METHODS: The experimental protocol first involved two ad libitum baseline days for HIGH (TFI, 3.21 l per 24 h; n=14) and LOW (TFI, 1.64 l per 24 h; n=14). During a controlled intervention (days 3-6), mineral water was the only beverage; HIGH consumed less than baseline (TFI, 2.00 l per 24 h), and LOW consumed more than baseline (TFI, 3.50 l per 24 h). During ad libitum recovery (day 7), TFI were 3.17 and 1.71 l per 24 h for HIGH and LOW, respectively. Duplicate Uosm (24 h collection) and Posm (morning) samples were analyzed on all days via freezing point depression osmometry. RESULTS: In the evaluation of relative water excess (Uosm:Posm<1.0), 11/13 values occurred for HIGH on days 1, 2 and 7; for LOW, 28/29 occurred on intervention days 3-6. Chi-squared analysis indicated that the treatment and Uosm:Posm were significantly associated (χ(2)1:0.001=23.5, P<0.001). Statistical regression analyses detected a strong, significant relationship between renal free-water clearance (FWC) and Uosm:Posm (r(2)=0.86, P<0.00001); this was not true for FWC and Posm (r(2)=0.00, P=0.40) because Posm values were stable across 7 days. CONCLUSIONS: These findings support the use of Uosm:Posm as a hydration biomarker.

Interpreting common hydration biomarkers on the basis of solute and water excretion.

BACKGROUND/OBJECTIVES: This investigation evaluated 12 hydration biomarkers, to determine which represent 24-h whole-body water balance (that is, measured as water retention or clearance (WR-C) by the kidneys). SUBJECTS/METHODS: Healthy males (n=59; body mass, 75.1±7.9 kg; height, 178±6 cm; age, 22±3 years; body mass index, 23.9±2.4 kg/m(2)) met with a registered dietitian each morning (days 1-11) to optimize completeness and accuracy of food and fluid records, then went about ordinary daily activities. These men visited the laboratory for blood samples and collected all urine produced on days 1, 3, 6, 9 and 12. The reference standard (WR-C) was calculated using 24-h urine volume, 24-h urine osmolality, and serum osmolality (single morning venous sample). RESULTS: Statistical regression analyses indicated that, among the 12 hydration biomarkers, only 24-h urine osmolality (r(2)=0.60, P<0.0001) and 24-h urine specific gravity (r(2)=0.52, P<0.0001) strongly predicted WR-C. The 24-h fluid intake, 24-h body mass change, 24-h urine color and 24-h urine volume were weak (P>0.05) predictors of WR-C, similar to serum osmolality and other single measurements (range of r(2) values, 0.19-0.0001). CONCLUSIONS: These observations of healthy, active young men demonstrate that WR-C is strongly related to the 24-h concentration of urine, which in turn reflects the excretion of total solids in the diet. Although morning urine assessments provided information about a single time point, 24-h urine osmolality and 24-h urine specific gravity were the best predictors of 24-h body water balance.

CpG stimulates protective immunity in Balb/cJ mice infected with larval Taenia crassiceps.

Balb/cJ mice infected in the peritoneal cavity with larval Taenia crassiceps fail to mount a protective immune response. In mice, inflammatory immune responses are believed to control larval reproduction, whereas antibody-mediated responses are believed to be permissive. In the present study, mice were treated with CpG-oligodeoxynucleotides (CpG) to determine whether stimulation of the innate inflammatory response would confer increased resistance to larval growth. Female mice treated with CpG displayed a decrease in mean parasite burden by 54%, while male mice displayed a 73% reduction. Moreover, 5 of 12 CpG-treated male mice completely eliminated all larvae by 9 wk post-infection. In contrast, no female animals were found to be infection free. CpG treatment induced an increase in the transcript levels of tumor necrosis factor-α and inducible nitric oxide synthase (iNOS) from splenocytes and resulted in elevated levels of the proinflammatory molecules monocyte chemotactic protein (MCP)-1, MCP-3, and interleukin-6 at the site of infection. Additionally, CpG administration induced the enhanced recruitment of neutrophils and macrophages to the site of infection. The finding that both neutrophils and macrophages were recruited in significantly higher numbers in the male host as compared to the female host may explain the increased level of protection realized in male animals in response to CpG treatment.

Changes in Thy1 gene expression associated with damaged retinal ganglion cells.

PURPOSE: The temporal series of molecular events that occur in dying retinal ganglion cells is poorly understood. We have examined the change in expression of a normally-expressed ganglion cell marker gene, Thy1, relative to the kinetics of cell loss caused by acute and chronic damaging stimuli. METHODS: For acute experiments, mice were subjected to optic nerve crush or intravitreal injections of N-methyl-D-aspartate (NMDA) to induce ganglion cell death. RNase protection analysis was used to quantify Thy1 mRNA levels from total retina RNA and in situ hybridization was used to monitor the pattern of Thy1 positive cells. Changes in Thy1 expression were compared to the time course of cell loss induced by each treatment. To induce elevated intraocular pressure (IOP), the episcleral veins of rats were injected with hypertonic saline, which scleroses Schlemm's Canal and the trabecular meshwork. Elevated IOP was monitored every day for 35 days after which the animals were sacrificed and the retinas harvested for quantitative RT-PCR or fixed for in situ hybridization studies. Evaluation of glaucomatous damage caused by elevated IOP was determined from histological sections of the optic nerves of all rat eyes. RESULTS: After optic nerve crush, Thy1 mRNA levels decreased within 24 h, although the number of expressing cells did not decline until 7 days. Both measures showed a loss of Thy1 well in advance of cell loss, which was detected by 2 weeks after surgery. This change in expression was not dependent on execution of the cell death program since a similar decrease was detected in Bax-/- ganglion cells, which are resistant to cell death induced by optic nerve crush. Thy1 mRNA levels and the number of expressing cells also decreased within 6 h after NMDA injection, in advance of cell loss, which was detected by 24 h. Similarly, elevated intraocular pressure was associated with a decrease in mRNA and expressing cells in a pressure-dependent manner. In moderately hypertensive rat eyes, the number of cells expressing Thy1 decreased before significant cell loss in the retina. Virtually no Thy1-expressing cells were detected in eyes with severe disease. CONCLUSIONS: Thy1 mRNA abundance and expressing cells, decreased in advance of detectable ganglion cell loss caused by three different modalities of damage. This change is independent of the committed step of cell death.

Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain.

The excitatory neurotransmitter, glutamate, is particularly important in the transmission of pain information in the nervous system through the activation of ionotropic and metabotropic glutamate receptors. A potent, subtype-selective antagonist of the metabotropic glutamate-5 (mGlu5) receptor, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), has now been discovered that has effective anti-hyperalgesic effects in models of inflammatory pain. MPEP did not affect rotarod locomotor performance, or normal responses to noxious mechanical or thermal stimulation in naïve rats. However, in models of inflammatory pain, systemic administration of MPEP produced effective reversal of mechanical hyperalgesia without affecting inflammatory oedema. In contrast to the non-steroidal anti-inflammatory drugs, indomethacin and diclofenac, the maximal anti-hyperalgesic effects of orally administered MPEP were observed without acute erosion of the gastric mucosa. In contrast to its effects in models of inflammatory pain, MPEP did not produce significant reversal of mechanical hyperalgesia in a rat model of neuropathic pain.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

Effect of general anesthetics on IOP in rats with experimental aqueous outflow obstruction.

PURPOSE: To determine the effect of several common general anesthetics on intraocular pressure (IOP) after experimental aqueous outflow obstruction in the rat. METHODS: A single episcleral vein injection of hypertonic saline was used to sclerose aqueous humor outflow pathways and produce elevated IOP in Brown Norway rats. Animals were housed in either standard lighting or a constant low-level light environment. Awake IOPs were determined using a TonoPen (Mentor, Norwell, MA) immediately before induction of anesthesia by either isoflurane, ketamine, or a mixture of injectable anesthetics (xylazine, ketamine, and acepromazine). For each anesthetic, IOPs were measured immediately after adequate sedation (time 0) and at 5-minute intervals, up to 20 minutes. RESULTS; Awake IOPs ranged from 18 to 52 mm Hg. All anesthetics resulted in a statistically significant (P: < 0.01) reduction in measured IOP at every duration of anesthesia when compared with the corresponding awake IOP. With increasing duration of anesthesia, measured IOP decreased approximately linearly for both the anesthetic mixture and isoflurane. However, with ketamine, IOP declined to 48% +/- 11% (standard lighting) and 60% +/- 7% (constant light) of awake levels at 5 minutes of anesthesia, where it remained stable. In fellow eyes, the SD of the mean IOP in animals under anesthesia was always greater than the corresponding SD of the awake mean. Anesthesia's effects in normal eyes and eyes with elevated IOP were indistinguishable. CONCLUSIONS: All anesthetics resulted in rapid and substantial decreases in IOP in all eyes and increased the interanimal variability in IOPs. Measurement of IOP in awake animals provides the most accurate documentation of pressure histories for rat glaucoma model studies.

Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats.

PURPOSE: To determine the diural intraocular pressure (IOP) response of Brown Norway rat eyes after sclerosis of the aqueous humor outflow pathways and its relationship to optic nerve damage. METHODS: Hypertonic saline was injected into a single episcleral vein in 17 animals and awake IOP measured in both the light and dark phases of the circadian cycle for 34 days. Mean IOP for light and dark phases during the experimental period were compared with the respective pressures of the uninjected fellow eyes. Optic nerve cross sections from each nerve were graded for injury by five independent masked observers. RESULTS: For fellow eyes, mean light- and dark-phase IOP was 21 +/- 1 and 31 +/- 1 mm Hg, respectively. For four experimental eyes, mean IOPs for both phases were not altered. Six eyes demonstrated significant mean IOP elevations only during the dark phase. Of these, five showed persistent, large circadian oscillations, and four had partial optic nerve lesions. The remaining seven eyes experienced significant IOP elevations during both phases, and all had extensive optic nerve damage. CONCLUSIONS: Episcleral vein injection of hypertonic saline is more likely to increase IOP during the dark phase than the light. This is consistent with aqueous outflow obstruction superimposed on a circadian rhythm of aqueous humor production. Because these periodic IOP elevations produced optic nerve lesions, both light- and dark-phase IOP determinations are necessary for accurate correlation of IOP history to optic nerve damage in animals housed in a light- dark environment.

Chronology of optic nerve head and retinal responses to elevated intraocular pressure.

PURPOSE: To determine the chronology of optic nerve head and retinal responses to elevated intraocular pressure (IOP). METHODS: After 1 to 39 days of unilaterally elevated IOP, experimental and fellow rat eyes were examined for morphology and immunohistochemical labeling alterations and for ganglion cell DNA fragmentation. RESULTS: Mean IOP for the experimental eyes was 36 +/- 8 mm Hg, an approximately 15-mm Hg elevation above normal values. By 7 days of pressure elevation above 40 mm Hg, endogenous immunostaining for brain-derived neurotrophic factor and neurotrophin 4/5 was absent from the nerve head and superior retina, whereas normal labeling was present in the inferior retina and distal optic nerve of these same eyes. These changes were preceded by a loss of gap junctional connexin43 labeling and astrocytic proliferation in the nerve head and by increased retinal ganglion cell layer apoptosis in the retina. Nerve head depletion of neurotrophins coincided with evidence of axonal degeneration, loss of astrocytic glial fibrillary acidic protein staining, and spread of collagen VI vascular immunolabeling. After longer durations at these same pressures, neurotrophin labeling returned to nerve head glia and scattered retinal ganglion cells. CONCLUSIONS: Optic nerve head and retinal responses, including the depletion of endogenous neurotrophins, are readily identified in the rat eye after experimental IOP elevation. However, the apparent chronology of these responses suggests that the withdrawal of neurotrophic support was not the only determinant of retinal ganglion cell apoptosis and axonal degeneration in response to pressure.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

Solution structure and dynamics of a designed hydrophobic core variant of ubiquitin.

BACKGROUND: The recent merger of computation and protein design has resulted in a burst of success in the generation of novel proteins with native-like properties. A critical component of this coupling between theory and experiment is a detailed analysis of the structures and stabilities of designed proteins to assess and improve the accuracy of design algorithms. RESULTS: Here we report the solution structure of a hydrophobic core variant of ubiquitin, referred to as 1D7, which was designed with the core-repacking algorithm ROC. As a measure of conformational specificity, we also present amide exchange protection factors and backbone and sidechain dynamics. The results indicate that 1D7 is similar to wild-type (WT) ubiquitin in backbone structure and degree of conformational specificity. We also observe a good correlation between experimentally determined sidechain structures and those predicted by ROC. However, evaluation of the core sidechain conformations indicates that, in general, 1D7 has more sidechains in less statistically favorable conformations than WT. CONCLUSIONS: Our results provide an explanation for the lower stability of 1D7 compared to WT, and suggest modifications to design algorithms that may improve the accuracy with which structure and stability are predicted. The results also demonstrate that core packing can affect conformational flexibility in subtle ways that are likely to be important for the design of function and protein-ligand interactions.

Microvasculature of the rat optic nerve head.

PURPOSE: To describe the arterial blood supply, capillary bed, and venous drainage of the rat optic nerve head. METHODS: Ocular microvascular castings from 6 Wistar rats were prepared by injection of epoxy resin through the common carotid arteries. After polymerization, tissues were digested with 6 M KOH, and the castings washed, dried, and coated for scanning electron microscopy. RESULTS: Immediately posterior to the globe, the ophthalmic artery trifurcates into the central retinal artery and two posterior ciliary arteries. The central retinal artery directly provides capillaries to the nerve fiber layer and only contributes to capillary beds in the neck of the nerve head. The remainder is supplied by branches of the posterior ciliary arteries that are analogous to the primate circle of Zinn-Haller. Arterioles arising from these branches supply the capillaries of the transitional, or laminar, region of the optic nerve head. These capillaries are continuous with those of the neck and retrobulbar optic nerve head. All optic nerve head capillaries drain into the central retinal vein and veins of the optic nerve sheath. A flat choroidal sinus communicates with the central retinal vein, the choriocapillaris, and with large veins of the optic nerve sheath. CONCLUSIONS: The microvasculature of the rat optic nerve head bears several similarities to that of the primate, with a centripetal blood supply from posterior ciliary arteries and drainage into the central retinal and optic nerve sheath veins. Association of nerve sheath veins with the choroid represents an important difference from the primate.

SIB-1757 and SIB-1893: selective, noncompetitive antagonists of metabotropic glutamate receptor type 5.

Cell lines expressing the human metabotropic glutamate receptor subtype 5a (hmGluR5a) and hmGluR1b were used as targets in an automated high-throughput screening (HTS) system that measures changes in intracellular Ca2+ ([Ca2+]i) using fluorescence detection. This functional screen was used to identify the mGluR5-selective antagonist, SIB-1757 [6-methyl-2-(phenylazo)-3-pyridinol], which inhibited the glutamate-induced [Ca2+]i responses at hmGluR5 with an IC50 of 0.37 microM compared with an IC50 of >100 microM at hmGluR1. Schild analysis demonstrated a noncompetitive mechanism of inhibition. Pharmacophore mapping was used to identify an additional compound, SIB-1893 [(E)-2-methyl-6-(2-phenylethenyl)pyridine], which was also shown to block glutamate-induced increases in [Ca2+]i at hmGluR5 with an IC50 of 0.29 microM compared with an IC50 of >100 microM at hmGluR1. SIB-1757 and SIB-1893 showed little or no activity when tested for agonist and antagonist activity at the other recombinant human mGluR subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and N-methyl-D-aspartate receptors. In rat neonatal brain slices, SIB-1757 and SIB-1893 inhibited (S)-3,5-dihydroxyphenylglycine (DHPG)-evoked inositol phosphate accumulation in hippocampus and striatum by 60% to 80%, with a potency similar to that observed on recombinant mGluR5. However, in the cerebellum, a brain region with low mGluR5 expression, SIB-1757 failed to inhibit DHPG-evoked inositol phosphate accumulation. In cultured rat cortical neurons, SIB-1757 and SIB-1893 largely inhibited DHPG-evoked [Ca2+]i signals, revealing a population of neurons that were less sensitive to SIB-1757 and SIB-1893. This is the first description of highly selective, noncompetitive mGluR5 antagonists. These compounds will be useful tools in evaluating the role of mGluR5 in normal physiology and in animal models of disease.

(R,S)-4-phosphonophenylglycine, a potent and selective group III metabotropic glutamate receptor agonist, is anticonvulsive and neuroprotective in vivo.

Group III metabotropic glutamate receptors (mGluRs) are thought to modulate neurotoxicity of excitatory amino acids, via mechanisms of presynaptic inhibition, such as regulation of neurotransmitter release. Here, we describe (R,S)-4-phosphonophenylglycine (PPG) as a novel, potent, and selective agonist for group III mGluRs. In recombinant cell lines expressing the human receptors hmGluR4a, hmGluR6, hmGluR7b, or hmGluR8a, EC50 values for (R,S)-PPG of 5.2 +/- 0.7 microM, 4.7 +/- 0.9 microM, 185 +/- 42 microM, and 0.2 +/- 0.1 microM, respectively, were measured. The compound showed EC50 and IC50 values of >/=200 microM at group I and II hmGluRs and was inactive at cloned human N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, and kainate receptors (>300 microM). On the other hand, it showed micromolar affinity for a Ca2+/Cl--dependent L-glutamate binding site in rat brain, similar to other phosphono-substituted amino acids like L-2-amino-4-phosphonobutyrate. In cultured cortical neurons, (R, S)-PPG provided protection against a toxic pulse of N-methyl-D-aspartate (EC50 = 12 microM), which was reversed by the group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate but not by the group II antagonist (2S)-alpha-ethylglutamate. Moreover, (R,S)-PPG protected against N-methyl-D-aspartate- and quinolinic acid-induced striatal lesions in rats and was anticonvulsive in the maximal electroshock model in mice. In contrast to the group III mGluR agonists L-2-amino-4-phosphonobutyrate and L-serine-O-phosphate, (R,S)-PPG showed no proconvulsive effects (2200 nmol i.c.v.). These data provide novel in vivo evidence for group III mGluRs as attractive targets for neuroprotective and anticonvulsive therapy. Also, (R,S)-PPG represents an attractive tool to analyze the roles of group III mGluRs in nervous system physiology and pathology.

Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine.

Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.