Lucia Ciranna

Activation of 5-HT7 serotonin receptors reverses metabotropic glutamate receptor-mediated synaptic plasticity in wild-type and Fmr1 knockout mice, a model of Fragile X syndrome.

BACKGROUND Fragile X syndrome (FXS) is a genetic cause of intellectual disability and autism. Fmr1 knockout (Fmr1 KO) mice, an animal model of FXS, exhibit spatial memory impairment and synapse malfunctioning in the hippocampus, with abnormal enhancement of long-term depression mediated by metabotropic glutamate receptors (mGluR-LTD). The neurotransmitter serotonin (5-HT) modulates hippocampal-dependent learning through serotonin 1A (5-HT1A) and serotonin 7 (5-HT7) receptors; the underlying mechanisms are unknown. METHODS We used electrophysiology to test the effects of 5-HT on mGluR-LTD in wild-type and Fmr1 KO mice and immunocytochemistry and biotinylation assay to study related changes of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) glutamate receptor surface expression. RESULTS Application of 5-HT or 8-OH-DPAT (a mixed 5-HT1A/5-HT7 agonist) reversed mGluR-LTD in hippocampal slices. Reversal of mGluR-LTD by 8-OH-DPAT persisted in the presence of the 5-HT1A receptor antagonist WAY-100635, was abolished by SB-269970 (5-HT7 receptor antagonist), and was mimicked by LP-211, a novel selective 5-HT7 receptor agonist. Consistently, 8-OH-DPAT decreased mGluR-mediated reduction of AMPA glutamate receptor 2 (GluR2) subunit surface expression in hippocampal slices and cultured hippocampal neurons, an effect mimicked by LP-211 and blocked by SB-269970. In Fmr1 KO mice, mGluR-LTD was abnormally enhanced; similarly to wild-type, 8-OH-DPAT reversed mGluR-LTD and decreased mGluR-induced reduction of surface AMPA receptors, an effect antagonized by SB-269970. CONCLUSIONS Serotonin 7 receptor activation reverses metabotropic glutamate receptor-induced AMPA receptor internalization and LTD both in wild-type and in Fmr1 KO mice, correcting excessive mGluR-LTD. Therefore, selective activation of 5-HT7 receptors may represent a novel strategy in the therapy of FXS.

Dysregulation of group-I metabotropic glutamate (mGlu) receptor mediated signalling in disorders associated with Intellectual Disability and Autism

Activation of group-I metabotropic glutamate receptors, mGlu1 and mGlu5, triggers a variety of signalling pathways in neurons and glial cells, which are differently implicated in synaptic plasticity. The earliest and much of key studies discovered abnormal mGlu5 receptor function in Fragile X syndrome (FXS) mouse models which then motivated more recent work that finds mGlu5 receptor dysfunction in related disorders such as intellectual disability (ID), obsessive-compulsive disorder (OCD) and autism. Therefore, mGlu1/5 receptor dysfunction may represent a common aetiology of these complex diseases. Furthermore, many studies have focused on dysregulation of mGlu5 signalling to synaptic protein synthesis. However, emerging evidence finds abnormal mGlu5 receptor interactions with its scaffolding proteins in FXS which results in mGlu5 receptor dysfunction and phenotypes independent of signalling to protein synthesis. Finally, both an increased and reduced mGlu5 functioning seem to be associated with ID and autism spectrum disorders, with important consequences for potential treatment of these developmental disorders.

5-HT 1A and 5-HT 7 receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission

We have studied the effects of 5‐HT1A and 5‐HT7 serotonin receptor activation in hippocampal CA3‐CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5‐HT or 8‐OH DPAT, a mixed 5‐HT1A/5‐HT7 receptor agonist, inhibited AMPA receptor‐mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5‐HT1A receptor agonist 8‐OH PIPAT and blocked by the 5‐HT1A antagonist NAN‐190. 8‐OH DPAT increased paired‐pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8‐OH DPAT enhanced EPSC amplitude but did not alter paired‐pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN‐190 and was blocked by the 5‐HT7 receptor antagonist SB‐269970. To confirm that EPSC enhancement was mediated by 5‐HT7 receptors, we used the compound LP‐44, which is considered a selective 5‐HT7 agonist. However, LP‐44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8‐OH DPAT. These effects were respectively antagonized by NAN‐190 and by SB‐269970, indicating that under our experimental condition LP‐44 behaved as a mixed agonist. 8‐OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5‐HT1A and 5‐HT7 receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5‐HT1A receptors inhibit CA3‐CA1 synaptic transmission acting both pre‐ and postsynaptically, whereas 5‐HT7 receptors enhance CA3‐CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal‐dependent cognitive functions. In this respect, we underline the need for new selective agonists of 5‐HT7 receptors.

Serotonin-evoked modifications of the neuronal firing rate in the superior vestibular nucleus: A microiontophoretic study in the rat

Microiontophoretic ejection (10-100 nA) of serotonin (5-hydroxytryptamine) into the superior vestibular nucleus induced modifications of the mean firing rate in 87% of the neurons examined. The responses to 5-hydroxytryptamine application were excitatory in 48% of the cells, inhibitory in 29%, and biphasic (inhibitory/excitatory) in the remaining 10%. The excited neurons were scattered throughout the nucleus; the units inhibited or characterized by biphasic responses were distinctly more numerous in the ventrolateral sector of the nucleus. The magnitude of both excitatory and inhibitory effects was dose-dependent. The excitatory responses to 5-hydroxytryptamine were blocked or greatly reduced by two 5-hydroxytryptamine antagonists, methysergide and ketanserin, or even reversed in many cases. Inhibitory responses were enhanced by simultaneous application of 5-hydroxytryptamine antagonists in half of the units studied. In the remaining units, ketanserin left the response unmodified, whereas methysergide reduced but never quite blocked it. The application of 5-methoxy-N,N- dimethyltryptamine, a 5-hydroxytryptamine agonist more effective on 5-hydroxytryptamine1 than on 5-hydroxytryptamine2 receptors, and of 8-hydroxy-2(di-n-propyl-amino) tetralin, a 5-hydroxytryptamine1A-specific agonist, induced a decrease in the firing rate which was unaffected by methysergide. These results support the hypothesis that 5-hydroxytryptamine exerts various functions throughout the superior vestibular nucleus by various receptors and that the inhibitory action is limited to an area of it.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT7 receptors as modulators of neuronal excitability, synaptic transmission and plasticity: physiological role and possible implications in autism spectrum disorders

Serotonin type 7 receptors (5-HT7) are expressed in several brain areas, regulate brain development, synaptic transmission and plasticity, and therefore are involved in various brain functions such as learning and memory. A number of studies suggest that 5-HT7 receptors could be potential pharmacotherapeutic target for cognitive disorders. Several abnormalities of serotonergic system have been described in patients with autism spectrum disorder (ASD), including abnormal activity of 5-HT transporter, altered blood and brain 5-HT levels, reduced 5-HT synthesis and altered expression of 5-HT receptors in the brain. A specific role for 5-HT7 receptors in ASD has not yet been demonstrated but some evidence implicates their possible involvement. We have recently shown that 5-HT7 receptor activation rescues hippocampal synaptic plasticity in a mouse model of Fragile X Syndrome, a monogenic cause of autism. Several other studies have shown that 5-HT7 receptors modulate behavioral flexibility, exploratory behavior, mood disorders and epilepsy, which include core and co-morbid symptoms of ASD. These findings further suggest an involvement of 5-HT7 receptors in ASD. Here, we review the physiological roles of 5-HT7 receptors and their implications in Fragile X Syndrome and other ASD.

Effects of noradrenaline on the firing rate of vestibular neurons

The effects of microiontophoretic noradrenaline on the firing rate of neurons located in the vestibular complex have been studied in anaesthetized rats. Eighty-five per cent of the neurons tested in all the vestibular nuclei modified their background firing rate upon noradrenaline application, generally by reducing it (86% of them). In few cases inhibitions were followed by a rebound. Responses were dose-dependent. No significant difference was found between vestibular neurons projecting to the spinal cord and those delivering their fibres to the oculomotor complex. Phentolamine, an alpha-adrenergic antagonist, blocked the noradrenaline-evoked inhibitions, whereas beta-adrenergic antagonist timolol was ineffective or enhanced them. Furthermore, responses were blocked by yohimbine, an alpha 2-adrenergic antagonist, and mimicked by clonidine, an alpha 2-adrenergic agonist, in the majority of neurons. In few cases prazosin, an alpha 1-adrenergic antagonist, was able to antagonize weak inhibitions and phenylephrine, an alpha 1-adrenergic agonist, to evoke an inhibitory effect blocked by prazosin. Isoproterenol, a beta-adrenergic agonist was totally ineffective on the neuronal firing rate. It is concluded that noradrenaline can modify the level of neuronal activity in the vestibular complex by acting mostly, but not exclusively, through alpha 2-adrenergic receptors. An influence of noradrenergic systems on the vestibular function by a direct action of noradrenaline inside the vestibular nuclei is proposed.

Modulation of AMPA receptor‐mediated ion current by pituitary adenylate cyclase‐activating polypeptide (PACAP) in CA1 pyramidal neurons from rat hippocampus

Pituitary adenylate cyclase‐activating polypeptide (PACAP), a neurotrophic and neuromodulatory peptide, was recently shown to enhance NMDA receptor‐mediated currents in the hippocampus (Macdonald, et al. 2005. J Neurosci 25:11374–11384). To check if PACAP might also modulate AMPA receptor function, we tested its effects on AMPA receptor‐mediated synaptic currents on CA1 pyramidal neurons, using the patch clamp technique on hippocampal slices. In the presence of the NMDA antagonist D‐AP5, PACAP (10 nM) reduced the amplitude of excitatory postsynaptic currents (EPSCs) evoked in CA1 pyramidal neurons by stimulation of Schaffer collaterals. Following a paired‐pulse stimulation protocol, the paired‐pulse ratio was unaffected in most neurons, suggesting that the AMPA‐mediated EPSC was modulated by PACAP mainly at a postsynaptic level. PACAP also modulated the currents induced on CA1 pyramidal neurons by applications of either glutamate or AMPA. The effects of PACAP were dose‐dependent: at a 0.5 nM dose, PACAP increased AMPA‐mediated current; such effect was blocked by PACAP 6–38, a selective antagonist of PAC1 receptors. The enhancement of AMPA‐mediated current by PACAP 0.5 nM was abolished when cAMPS‐Rp, a PKA inhibitor, was added to the intracellular solution. At a 10 nM concentration, PACAP reduced AMPA‐mediated current; such effect was not blocked by PACAP 6–38. The inhibitory effect of 10 nM PACAP was mimicked by Bay 55–9837 (a selective agonist of VPAC2 receptors), persisted in the presence of intracellular BAPTA and was abolished by intracellular cAMPS‐Rp. Stimulation‐evoked EPSCs in CA1 neurons were significantly reduced following application of the PAC1 antagonist PACAP 6–38; this result indicates that PAC1 receptors in the CA1 region are tonically activated by endogenous PACAP and enhance CA3‐CA1 synaptic transmission. Our results show that PACAP differentially modulates AMPA receptor‐mediated current in CA1 pyramidal neurons by activation of PAC1 and VPAC2 receptors, both involving the cAMP/PKA pathway; the functional significance will be discussed in light of the multiple effects exerted by PACAP on the CA3‐CA1 synapse at different levels.

Computational Studies to Discover a New NR2B/NMDA Receptor Antagonist and Evaluation of Pharmacological Profile

The ionotropic glutamate NMDA/NR2B receptor and its interaction with ifenprodil‐like noncompetitive ligands were investigated by a combined ligand‐based and target‐based approach. First, we generated 3D pharmacophore hypotheses and identified common chemical features that are shared by a training set of well‐known NR2B antagonists. The binding mode of the most representative ligand was also studied by molecular docking. Because the docking results and the suggested 3D pharmacophore model were in good agreement, we obtained new information about the NR2B ifenprodil site. The best pharmacophoric hypothesis was used as a query for in silico screening; this allowed the identification of new “hit”. We synthesized “hit‐compound” analogues, and some of the molecules showed significant activity both in binding and functional assay as well as in vivo anticonvulsant efficacy in DBA/2 mice. The most active derivatives also exhibited neuroprotective effects against glutamate‐induced toxicity in HCN‐1A cells.

Development of 3-substituted-1H-indole derivatives as NR2B/NMDA receptor antagonists.

A combined ligand-based and structure-based approach has previously allowed us to identify NR2B/NMDA receptor antagonists containing indole scaffold. In order to further explore the main structure activity relationships of this class of derivatives we herein report the design, synthesis and biological evaluation of new analogues. Some derivatives demonstrated to produce significant anticonvulsant properties and NMDA antagonism. The most active of them (3d) showed NR2B binding affinity equipotent to that of ifenprodil. These results were also corroborated by computational studies.

5-Hydroxytryptamine modifies neuronal responses to glutamate in the red nucleus of the rat

Abstract The effects of 5-hydroxytryptamine (5-HT) on the responses of red nucleus (RN) neurones to glutamate (glu) and its agonists were studied using a microiontophoretic technique in anaesthetised rats. Extracellular unitary recordings of RN neuronal activity showed that 5-HT application induced a significant and reversible depression of glu-evoked excitations in 85% of the RN units tested. This effect was independent of the action of the amine on background firing, which appeared enhanced in the majority of cases but was either depressed or uninfluenced in other cases. Microiontophoretic 5-HT also depressed the excitatory responses evoked in RN neurones by electrical stimulation of sensorimotor cortex. Methysergide application, which prevented the enhancing effects of 5-HT on the background firing, was scarcely effective in antagonising the depression of glu responses. In contrast, the serotonergic effects on the glu responses were reduced by the iontophoretically applied antagonist of 5-HT1A receptors, NAN-190. Microiontophoretic 5-HT was also able to influence the neuronal responses evoked by glu agonists quisqualate (quis) and N-methyl-d-aspartate (NMDA), acting on non-NMDA and NMDA receptors respectively. In fact 5-HT depressed quis-evoked excitations and induced mixed effects on NMDA responses, which were reduced in 45%, enhanced in 34% and unmodified in 21% of the units tested. These results suggest that 5-HT is able to modulate the motor glutamatergic input to RN by acting mostly on non-NMDA receptors. The modulation of non-NMDA and NMDA receptors by 5-HT in the RN appears significant and its functional meaning is discussed.