Melvyn Baez

Metabotropic glutamate 5 receptor antagonism is associated with antidepressant-like effects in mice

Antidepressant-like effects of metabotropic glutamate (mGlu)5 receptor antagonists have been reported previously. We now provide definitive identification of mGlu5 receptors as a target for these effects through the combined use of selective antagonists and mice with targeted deletion of the mGlu5 protein. In these experiments, the mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and the more selective and metabolically stable analog 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) decreased immobility in the mouse forced swim test, a test predictive of antidepressant efficacy in humans. mGlu5 receptor knockout mice had a phenotype in the forced swim test that was congruent with the effects of receptor blockade; mGlu5 receptor knockout mice were significantly less immobile than their wild-type counterparts. Consistent with mGlu5 receptor mediation of the antidepressant-like effects of MPEP, the effects of MPEP were not observed in mGlu5 receptor knockout mice, whereas comparable effects of the tricyclic antidepressant imiprimine remained active in the mutant mice. MPEP and imiprimine resulted in a synergistic antidepressant-like effect in the forced swim test. The drug interaction was not likely because of increased levels of drugs in the brain, suggesting a pharmacodynamic interaction of mGlu5 and monoaminergic systems in this effect. Thus, the present findings substantiate the hypothesis that mGlu5 receptor antagonism is associated with antidepressant-like effects. This mechanism may not only provide a novel approach to the therapeutic management of depressive disorders but also may be useful in the augmentation of effects of traditional antidepressant agents.

Clusterin (Apo J) Protects Against In Vitro Amyloid-β(1–40) Neurotoxicity

Abstract: Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimers disease (AD), where it is found associated with amyloid‐β (Aβ) plaques. Clusterin also coprecipitates with Aβ from CSF, suggesting a physiological interaction with Aβ. Given this interaction with Aβ, the goal of this study was to determine whether clusterin could modulate Aβ neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC‐9, a full‐length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose‐dependent neuroprotection against Aβ(1–40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of Aβ(1–40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid‐β plaque pathology.

Increased anxiety-related behavior in mice deficient for metabotropic glutamate 8 (mGlu8) receptor.

Pre-synaptic metabotropic glutamate (mGlu) receptors modulate neuronal excitability by controlling glutamate and gamma-aminobutyric acid (GABA) release. The mGlu8 receptor is predominantly found in pre-synaptic terminals and its expression is highly restricted. To study the role of this receptor, mGlu8 receptor-deficient mice were generated. Here we report that naïve mGlu8 receptor-deficient mice showed increased anxiety-related behavior in the elevated plus maze in low illumination conditions (red light). Open arm avoidance and risk assessment behavior were both significantly increased in mutant mice. Increased stressfulness of the testing conditions abolished this behavioral difference. Fluorescent light or prior restraint stress decreased the open arm activity of wild-type mice, while the open arm activity of mutant mice was essentially unaffected, leading to similar values in both strains. The total number of arm entries or closed arm entries was not significantly different between strains, indicating that the lack of mGlu8 receptor does not affect locomotor activity. No gross behavioral changes, or changes in the function of the autonomic nervous system or somatomotor systems were observed in mutant mice. Moreover, no significant differences in seizure susceptibility were detected between strains. Our results suggest that mGlu8 receptor may play a role in responses to novel stressful environment.

Receptor subtype and density determine the coupling repertoire of the 5-ht2 receptor subfamily

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.

Activation of Erk mitogen-activated protein kinase proteins by vascular serotonin receptors.

We previously demonstrated that 5-hydroxytryptamine 2A (5-HT 2A ) receptor–mediated rat arterial contraction was dependent on activation of tyrosine kinases, including mitogen-activated protein kinase (MAPK) kinase. In the current study, we examined arterial smooth muscle for the presence of serotonin (5-hydroxytryptamine, 5-HT) 5-HT 1B , 5-HT 1D , 5-HT 1F , 5-HT 2A , 5-HT 2B , and 5-HT 7 receptor mRNA and hypothesized that, if present, activation of these receptors would stimulate the extracellular signal–regulated kinase (Erk) MAPK pathway and an Erk MAPK–dependent contraction. RT-PCR analyses of rat aortic smooth muscle cells, cultured and fresh, indicated the presence of 5-HT 1B , 5-HT 1D , 5-HT 1F , 5-HT 2A , 5-HT 2B , and 5-HT 7 receptor mRNA. The 5-HT 1B agonists RU24969 and CGS12066B, 5-HT 1B/1D/1F receptor agonist sumatriptan, and 5-HT 2B receptor agonist BW723C86 (10 −9 –10 −4M) did not contract the aorta, nor did the 5-HT 7 receptor antagonist LY215840 leftward shift 5-HT–induced contraction. The 5-HT 1E/1F receptor agonist BRL54443 induced contraction, but this was abolished by the 5-HT 2A/2C receptor antagonist ketanserin (10 n M); contraction was not observed with a different 5-HT 1F receptor agonist, LY344864. 5-HT and &agr;-methyl-5-HT produced a concentration-dependent increase in Erk MAPK activity in cultured aortic smooth muscle cells and in aorta contracted with these agonists. All other agonists were inactive; a high concentration of BRL54443 (10 &mgr;M) stimulated Erk MAPK activation (150% basal). Thus, while mRNA and possibly protein for multiple 5-HT receptors are present in aortic smooth muscle, only the 5-HT 2A receptor plays a significant role in directly modulating contractility and activating the Erk MAPK pathway.

Modulation of lateral perforant path excitatory responses by metabotropic glutamate 8 (mGlu8) receptors

The contribution of metabotropic glutamate 8 (mGlu8) receptors to modulation of medial and lateral perforant path (MPP and LPP) inputs to the dentate gyrus was investigated using electrophysiological recording of field excitatory postsynaptic potentials (fEPSPs) from hippocampal slices taken from wild-type and mGlu8 receptor knockout animals. Application of the selective group III mGlu receptor agonist, L-AP4 (1-100 microM), reduced fEPSPs evoked by LPP, but not MPP stimulation in wild-type slices in a concentration-dependent manner (EC(50) = 4.7 microM). The selective mGlu8 receptor agonist, DCPG (1-30 microM) also suppressed LPP fEPSPs with an EC(50) value of 3.1 microM. The L-AP4-induced reduction in LPP fEPSPs could be blocked by the group III antagonist, MSOP (100 microM) in wild-type slices and was eliminated in mGlu8 receptor-deficient slices. Additional experiments showed that MPP fEPSPs were suppressed by the group II agonist, LY379268 (0.01-3 microM) in control slices (EC(50) = 153.1 nM); an effect that was not altered in mGlu8 receptor knockout slices (EC(50) = 153.8 nM). In addition, LY379268 had little effect on fEPSPs evoked by LPP stimulation in mGlu8 receptor-deficient slices. In conjunction with recent receptor localization studies, these results suggest that the mGlu8 receptors serve as autoreceptors on LPP afferents to the dentate gyrus.

Systemic administration of the potent mGlu8 receptor agonist (S)-3,4-DCPG induces c-Fos in stress-related brain regions in wild-type, but not mGlu8 receptor knockout mice

The effect of a novel and potent metabotropic glutamate 8 (mGlu8) receptor agonist, (S)-3,4-dicarboxyphenylglycine (DCPG), was studied in vivo in mouse brain. c-Fos expression was used as a marker of neuronal activity in specific brain regions 2 h after systemic (S)-3,4-DCPG (3-100 mg/kg, i.p.). The selectivity of (S)-3,4-DCPG on mGlu8 receptors was determined in mGlu8 receptor knockout mice. In wild-type mice, (S)-3,4-DCPG (100 mg/kg) significantly increased c-Fos expression in several stress-related brain regions: paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, lateral parabrachial nucleus and locus coeruleus. In the central nucleus of the amgydala, more than 92% of c-Fos positive neurons were identified as GABAergic inhibitory neurons after (S)-3,4-DCPG. Moreover, (S)-3,4-DCPG significantly induced c-Fos in the superficial gray layer of the superior colliculus, a central visual region. c-Fos expression was unchanged by (S)-3,4-DCPG in mGlu8 receptor knockout mice. Our results indicate that systemic (S)-3,4-DCPG alters neuronal excitability in specific brain regions via mGlu8 receptor. The prominent activation of stress areas suggests a role for mGlu8 receptors in the central integration of stress responses. Furthermore, our results indicate that systemic (S)-3,4-DCPG can be used as a tool to explore behavioral and cellular consequences of mGlu8 receptor activation.

Constitutive deletion of the serotonin-7 (5-HT7) receptor decreases electrical and chemical seizure thresholds

The localization of serotonin-7 (5-HT(7)) receptors and the biological activity of ligands have suggested that 5-HT(7) receptors might be involved in pain, migraine, epilepsy, anxiety, depression, memory, and sleep. In the present study, the potential involvement of 5-HT(7) receptors in epilepsy and other seizure disorders was assessed by comparing the seizures produced by three types of electrical stimulation and three chemical convulsants in 5-HT(7) receptor-deficient (knockout, KO) mice to those seizures observed in wild-type (WT) mice. Thresholds for producing electroshock-induced clonic seizures did not differ between KO versus WT mice. However, thresholds for producing electroshock-induced tonic seizures were significantly lower in KO than in WT mice. Seizures produced by pentylenetetrazole (PTZ, a GABA(A) receptor antagonist), N-methyl-d-aspartate (NMDA, an agonist at NMDA-type glutamate receptors), and cocaine (an inhibitor of monoamine uptake) were also studied. PTZ was more potent in inducing seizures in 5-HT(7) KO mice than in wild-type mice. Likewise, cocaine was more potent in inducing seizures in 5-HT(7) KO than in WT mice; moreover, death resulted from cocaine administration in 5-HT(7) KO mice but not in WT mice. There was a similar trend for NMDA that did not reach statistical significance. The present findings point to the potential for a generalized reduction in seizure threshold with constitutive deletion of the 5-HT(7) receptor gene. Since seizures have not been reported with pharmacological blockade of the receptor, the findings suggest that adaptive changes may play a role in the low seizure thresholds in these mice. In addition, the data suggest that the lower thresholds for seizures produced by diverse mechanisms should be taken into account when interpreting other aspects of the phenotype and behavioral pharmacology of this mouse.

Serotonin-induced contraction in canine coronary artery and saphenous vein: Role of a 5-HT1D-like receptor

The identity of the serotonin (5-HT) receptor(s) that mediate(s) contraction in canine coronary artery and saphenous vein remains controversial. Ring segments of endothelium-denuded coronary artery and helical strips of saphenous vein were suspended in organ chambers for measurement of isometric force. 5-HT, alpha Me-5-HT and sumatriptan contracted both coronary artery and saphenous vein and the non-selective 5-HT receptor antagonist 1-naphthylpiperazine (100nM) blocked 5-HT- and sumatriptan-induced contraction in both tissues. The agonist rank order potency for contraction (5-HT > sumatriptan > alpha Me5-HT > 5-MeOT > 5-MeT) was similar in both tissues and was consistent with that for a 5-HT1D receptor. Oligonucleotide primers specific for the 5-HT1D receptor sequence were designed for use in a polymerase chain reaction (PCR). cDNA derived from total RNA or mRNA from canine tissues was used in the PCR. PCR resulted in the amplification of a 632 base pair sequence in both canine coronary artery and saphenous vein; consistent with that expected for the 5-HT1D receptor. Southern blot analysis, with an oligonucleotide probe internal to the sequence amplified by the PCR primers, confirmed that the sequence amplified by PCR was the 5-HT1D receptor. Thus, the 5-HT1D receptor is expressed in canine coronary artery and saphenous vein and taken together with the pharmacological data, supports the possibility that a 5-HT1D-like receptor mediates contraction in these two tissues.

[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor

Abstract In previous reports, [3H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT2B receptors. 5-HT, the native agonist for the 5-HT2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [3H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (Ki human = 14.3 ± 1.2 nM, compared to Ki rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [3H]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [3H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α2-adrenergic ligands, it was determined that there was a component of [3H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α2-adrenergic receptor. The α2-adrenergic ligand efaroxan was found to block [3H]rauwolscine binding to the α2-adrenergic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [3H]rauwolscine labeled a single population of binding sites, Kd = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [3H]rauwolscine labeled 783 ± 10 fmol of 5-HT2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT2B receptors/mg of protein for [3H]5-HT binding. At 0° C, where the conditions for [3H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT2B receptor, [3H]rauwolscine (Bmax = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [3H]5-HT (Bmax = 615 ± 34 fmol/mg). The affinity of [3H]rauwolscine for the human 5-HT2B receptor at 0° C did not change, Kd = 4.93 ± 1.27 nM, while that for [3H]5-HT increased greatly (Kd at 37° C = 7.76 ± 1.06 nM; Kd at 0° C = 0.0735 ± 0.0081 nM). When using [3H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT2B receptor when [3H]rauwolscine was the radioligand. Typically, ∼ 85% of [3H]rauwolscine binding was specific binding. These studies display the usefulness of [3H]rauwolscine as an antagonist radioligand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT2B receptor.