Mauro Corsi

Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice

Both ionotropic and metabotropic glutamate receptors (mGluRs) are involved in the behavioral effects of pyschostimulants; however, the specific contributions of individual mGluR subtypes remain unknown. Here we show that mice lacking the mGluR5 gene do not self-administer cocaine, and show no increased locomotor activity following cocaine treatment, despite showing cocaine-induced increases in nucleus accumbens (NAcc) dopamine (DA) levels similar to wild-type (WT) mice. These results demonstrate a significant contribution of mGlu5 receptors to the behavioral effects of cocaine, and suggest that they may be involved in cocaine addiction.

Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain

Substance P exerts its various biochemical effects mainly via interactions through neurokinin‐1 receptors (NK1). Recently, the NK1 receptor has attracted considerable interest for its possible role in a variety of psychiatric disorders including depression and anxiety. However, little is known regarding the anatomical distribution of NK1 in the human central nervous system (CNS). Riboprobe in situ hybridization, quantitative PCR and in vitro autoradiography were performed. Highest NK1 mRNA levels were localized in the locus coeruleus and ventral striatum, while moderate hybridization signals were observed in the cerebral cortex (most abundant in the visual cortex), hippocampus and different amygdaloid nuclei. Very low levels of the NK1 mRNA were detected in the cerebellum and thalamus. In view of the existence of a long and short isoform of the NK1 receptor, it was of interest to assess whether there was a differential distribution of the two splice variants in the human CNS and peripheral tissues. A quantitative TaqMan PCR analysis showed that the long NK1 isoform was the most prevalent throughout the human brain, while in peripheral tissues the truncated form was the most represented. 3H‐Substance P autoradiography revealed a good correlation between receptor binding sites and NK1 mRNA expression throughout the brain, with the highest levels of binding in the locus coeruleus. These results provide the anatomical evidence that the NK1 receptors have a strong association with neuronal systems relevant to mood regulation and stress in the human brain, but do not suggest a region‐specific role of the two isoforms in the CNS.

The Use of Knock-Out Mice Unravels Distinct Roles for mGlu2 and mGlu3 Metabotropic Glutamate Receptors in Mechanisms of Neurodegeneration/Neuroprotection

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (−)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2−/−, or mGlu3−/− mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2−/− mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2−/− mice but not in mGlu3−/− mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

Activation of the extracellular signal-regulated kinase 2 by metabotropic glutamate receptors

Activation of metabotropic glutamate receptors (mGluRs) leads to modulation of a variety of second messenger pathways probably including the mitogen‐activated protein kinase (MAPK) extracellular signal‐regulated protein kinases (ERK). MAPK play a key role in the control of cellular responses to changes in the external environment by regulating transcriptional activity and the phosphorylation state of several cytoplasmic targets. In this study, Chinese hamster ovary (CHO) cells permanently transfected with rat mGluR1a, mGluR2 and mGluR4 were employed as a model to examine the activation of MAPK by glutamate through mGluRs. All three mGluR subtypes rapidly stimulated ERK activation. In particular, mGluR1a and mGluR2 preferentially mediated phosphorylation and activation of ERK2 in a pertussis toxin (PTX)‐sensitive and concentration‐dependent manner. The activation was blocked completely by pretreatment with the antagonist (rs)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) or with the MEK inhibitor PD098059. Furthermore, mGluR1a‐mediated ERK activation was suppressed by the depletion of endogenous protein kinase C (PKC) activity and by the PKC inhibitors staurosporine and calphostin C, but not chelerythrine. When cAMP was elevated in mGluR2‐expressing cells, by forskolin or dibutyryl‐cAMP, slight elevation of ERK activity was observed. However, glutamate‐stimulated ERK activation remained unaffected. In these cells, the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin produced a significant, albeit only partial, inhibition of mGluR2‐mediated ERK activation. These findings raise the possibility of a MAPK cascade involvement in glutamate‐dependent neuronal plasticity mediated through stimulation of mGluRs.

CLONING AND CHARACTERIZATION OF ALTERNATIVE MRNA FORMS FOR THE RAT METABOTROPIC GLUTAMATE RECEPTORS MGLUR7 AND MGLUR8

Novel mRNA isoforms for two members of the group III metabotropic glutamate receptors (mGluRs), called mGluR7b and mGluR8b, were identified from rat brain cerebral cortex and hippocampus. In both cases, the alternative splicing is generated by a similar out‐of‐frame insertion in the carboxyl‐terminus that results in the replacement of the last 16 amino acids of mGluR7 and mGluR8 by 23 and 16 different amino acids, respectively. Distribution analysis for mGluR7 and mGluR8 isoforms revealed that the two splice variants are generally coexpressed in the same brain areas. The few exceptions were the olfactory bulb, in which only the mGluR7a form could be detected by reverse transcription–polymerase chain reaction, and the lateral reticular and ambiguus nuclei, which showed only mGluR8a labelling. Despite expression in the same regions, different mRNA abundance for the two variants of each receptor were observed. When transiently coexpressed in HEK 293 cells with the phospholipase C‐activating chimeric Gαqi9‐G‐protein, the a and b forms for both receptor subtypes showed a similar pharmacological profile. The rank order of potencies for both was: dl‐amino‐4‐phosphonobutyrate > l‐serine‐O‐phosphate > glutamate. However, the agonist potencies were significantly higher for mGluR8a, b compared with mGluR7a,b. In Xenopus oocytes, glutamate evoked currents only with mGluR8 when coexpressed with Kir 3.1 and 3.4. Glutamate‐induced currents were antagonized by the group II/III antagonist (RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine. In conclusion, the two isoforms of each receptor have identical pharmacological profiles when expressed in heterologous systems, despite structural differences in the carboxyl‐terminal domains.

The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca2+ channels encoded by the CaV3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three CaV3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. CaV3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic CaV3.3−/− mouse line and demonstrate that CaV3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of CaV3.3 channels prevented oscillatory bursting in the low-frequency (4–10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing CaV3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in CaV3.3−/− mice were <30% compared with those in WT mice, and the remaining current, carried by CaV3.2 channels, generated dendritic [Ca2+]i signals insufficient to provoke oscillatory bursting that arises from interplay with Ca2+-dependent small conductance-type 2 K+ channels. Finally, naturally sleeping CaV3.3−/− mice showed a selective reduction in the power density of the σ frequency band (10–12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for CaV3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

Structure-Activity Studies on Neuropeptide S IDENTIFICATION OF THE AMINO ACID RESIDUES CRUCIAL FOR RECEPTOR ACTIVATION

Neuropeptide S (NPS) has been recently recognized as the endogenous ligand for the previous orphan G-protein-coupled receptor GPR154, now referred to as the NPS receptor (NPSR). The NPS-NPSR receptor system regulates important biological functions such as sleeping/wakening, locomotion, anxiety, and food intake. To collect information on the mechanisms of interaction between NPS and its receptor, a classical structure-activity relationship study was performed. Human (h) NPS derivatives obtained by Ala and d-scan and N- and C-terminal truncation were assessed for their ability to stimulate calcium release in HEK293 cells expressing the human recombinant NPSR. The results of this study indicate that (i) the effect of hNPS is mimicked by the fragment hNPS-(1–10); (ii) Phe2, Arg3, and Asn4 are crucial for biological activity; (iii) the sequence Thr8-Gly9-Met10 is important for receptor activation, although with non-stringent chemical requirements; and (iv) the sequence Val6-Gly7 acts as a hinge region between the two above-mentioned domains. However, the stimulatory effect of hNPS given intracerebroventricularly on mouse locomotor activity was not fully mimicked by hNPS-(1–10), suggesting that the C-terminal region of the peptide maintains importance for in vivo activity. In conclusion, this study identified the amino acid residues of this peptide most important for receptor activation.

Functional Magnetic Resonance Imaging Reveals Different Neural Substrates for the Effects of Orexin-1 and Orexin-2 Receptor Antagonists

Orexins are neuro-modulatory peptides involved in the control of diverse physiological functions through interaction with two receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Recent evidence in pre-clinical models points toward a putative dichotomic role of the two receptors, with OX2R predominantly involved in the regulation of the sleep/wake cycle and arousal, and the OX1R being more specifically involved in reward processing and motivated behaviour. However, the specific neural substrates underlying these distinct processes in the rat brain remain to be elucidated. Here we used functional magnetic resonance imaging (fMRI) in the rat to map the modulatory effect of selective OXR blockade on the functional response produced by D-amphetamine, a psychostimulant and arousing drug that stimulates orexigenic activity. OXR blockade was produced by GSK1059865 and JNJ1037049, two novel OX1R and OX2R antagonists with unprecedented selectivity at the counter receptor type. Both drugs inhibited the functional response to D-amphetamine albeit with distinct neuroanatomical patterns: GSK1059865 focally modulated functional responses in striatal terminals, whereas JNJ1037049 induced a widespread pattern of attenuation characterised by a prominent cortical involvement. At the same doses tested in the fMRI study, JNJ1037049 exhibited robust hypnotic properties, while GSK1059865 failed to display significant sleep-promoting effects, but significantly reduced drug-seeking behaviour in cocaine-induced conditioned place preference. Collectively, these findings highlight an essential contribution of the OX2R in modulating cortical activity and arousal, an effect that is consistent with the robust hypnotic effect exhibited by JNJ1037049. The subcortical and striatal pattern observed with GSK1059865 represent a possible neurofunctional correlate for the modulatory role of OX1R in controlling reward-processing and goal-oriented behaviours in the rat.

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder

Cumulative concentration‐response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2α (0.01–30 μM) and to the thromboxane A2 (TXA2) receptor agonist U‐46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2α>U‐46619>PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM. The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U‐46619‐induced contractions with an apparent pKB value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF2α and it was a non‐surmountable antagonist of PGE2 (apparent pKB of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U‐46619 (apparent pKB value of 5.88±0.04; n=4). Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U‐46619 (0.01–3 μM) potentiated the twitch contraction in a dose‐dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pKB of 8.54±0.14 (n=4 for each antagonist concentration). PGF2α in the range 0.01–10 μM (n=7), but not PGE2 and PGE1 (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM‐induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5). Cumulative additions of U‐46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U‐46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2). We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2α are also present. All these receptors are probably located post‐junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE2 seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA2 and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

Pharmacological analysis of 5-hydroxytryptamine effects on electrically stimulated human isolated urinary bladder

1 5‐Hydroxytryptamine (5‐HT) is able to potentiate the contractions induced by electrical field stimulation of pieces of human isolated urinary bladder. On the basis of available selective 5‐HT agonists and antagonists, we have further investigated the receptors involved and their site of action. 2 5‐HT produced a concentration‐dependent increase of the contractile response to electrical field stimulation from 0.1 nm to μm. At higher concentrations (up to 100 μm) the effect decreased. These activities were mimicked by a variety of 5‐HT agonists, for which the following rank order of potency was found: 5‐HT > α‐methyl 5‐HT > 5‐methoxytryptamine > 5‐carboxamidotryptamine > 2‐methyl 5‐HT ≫ GR 43175. In addition the gastro‐prokinetics agents metoclopramide, cisapride and the 5‐HT3 antagonist ICS 205–930 behaved as 5‐HT agonists, their EC50 values being 2.3, 0.3, and 0.5 (μm) respectively. 3 The 5‐HT potentiating effect was resistant to antagonism by ondansetron (1 μm) and cyanopindolol (1 μm), selective 5‐HT3 and 5‐HT1A/1B antagonists respectively. The 5‐HT2 antagonists ketanserin (1 μm), spiperone (1 μm) and methysergide (1 μm) also showed a weak inhibitory activity. Methiothepin (0.1–1 μm) antagonized only the inhibitory effect of 5‐HT. Metoclopramide (0.1–1 μm), cisapride (0.01–0.1 μm) and ICS 205–930 (0.3–3 μm) all produced a rightward displacement of the 5‐HT response curve with concomitant reduction of the maximum response. The pA2 values calculated were 7.4, 8.5 and 7.0 respectively. The antagonism of metoclopramide was receptor specific and was not apparently related to interactions with dopaminergic activity since domperidone showed no antagonism of 5‐HT, and metoclopramide, itself, did not antagonize the potentiating effect of prostaglandin F2α. 4 The receptor involved in the potentiating effect of 5‐HT may be located prejunctionally because 5‐HT did not potentiate responses to acetylcholine (ACh) or electrical field stimulation with the parameters of direct muscle excitation. Also, since the 5‐HT potentiating effect was blocked by atropine, it may be attributed to a release of ACh. 5 This study suggests that in the human urinary bladder 5‐HT causes two opposite effects on the contractile response to electrical field stimulation. A potentiating effect at low concentrations due to an interaction with an atypical receptor, different from the classical 5‐HT1, 5‐HT2 or 5‐HT3 subtypes and an inhibitory effect at greater concentrations probably due to an interaction with 5‐HT1‐like receptors. The possibility that this atypical receptor possesses some characteristics of those found in other isolated preparations like guinea‐pig ileum, rat oesophagus and mouse embryo colliculi neurones is discussed.