Maurizio Cammalleri

Time-restricted role for dendritic activation of the mTOR-p70S6K pathway in the induction of late-phase long-term potentiation in the CA1

Mammalian target of rapamycin (mTOR) is a key regulator of translational capacity. The mTOR inhibitor rapamycin can prevent forms of protein synthesis-dependent synaptic plasticity such as long-term facilitation in Aplysia and late-phase long-term potentiation (L-LTP) in the hippocampal CA1 region of rodents. In the latter model, two issues remain to be addressed: defining the L-LTP phase sensitive to rapamycin and identifying the site of rapamycin-sensitive protein synthesis. Here, we show that L-LTP is sensitive to application of rapamycin only during the induction paradigm, whereas rapamycin application after the establishment of L-LTP was ineffective. Second, we observed that Thr-389-phosphorylated p70 S6 kinase (p70S6K), the main active phosphoform of the mTOR effector p70S6K, was induced in an N-methyl-d-aspartateand phosphatidylinositol 3-kinase-dependent manner throughout the dendrites but not in the cell bodies of CA1 neurons in hippocampal slices after L-LTP induction. A similar dendrite-wide activation of p70S6K was induced in primary hippocampal neurons by depolarization with KCL or glutamate. In primary hippocampal neurons, the sites of dendritic activation of p70S6K appeared as discrete compartments along dendritic shafts like the hotspots for fast dendritic translation. Conversely, only a subset of dendritic spines also displayed activated p70S6K. Taken together, the present data suggest that the N-methyl-d-aspartate-, phosphatidylinositol 3-kinase-dependent dendritic activation of the mTOR-p70S6K pathway is necessary for the induction phase of protein synthesis-dependent synaptic plasticity. Newly synthesized proteins in dendritic shafts could be targeted selectively to activity-tagged synapses. Thus, coordinated activation of dendrite-wide translation and synaptic-specific activation is likely to be necessary for long-term synaptic plasticity.

Role of the adrenergic system in a mouse model of oxygen-induced retinopathy: antiangiogenic effects of beta-adrenoreceptor blockade.

PURPOSE Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). In OIR mice, this study determined whether blockade of β-adrenergic receptors (β-ARs) with propranolol influences retinal levels of proangiogenic factors, retinal vascularization, and blood-retinal barrier (BRB) breakdown. METHODS Propranolol was administered subcutaneously and picropodophyllin (PPP) intraperitoneally. Intravitreal injections of vascular endothelial growth factor (VEGF) were performed. Messengers of β-ARs, VEGF, its receptors, IGF-1 and IGF-1R were measured with quantitative RT-PCR. VEGF content was determined with ELISA. β-ARs, hypoxia-inducible factor (HIF)-1α, occludin, and albumin were measured with Western blot. Retinal localization of β3-ARs was determined by immunohistochemistry. Retinopathy was assessed by scoring fluorescein-perfused retinas, and plasma extravasation was visualized by Evans blue dye. RESULTS Hypoxia did not influence β-AR expression, except that it increased β3-AR protein with dense β3-AR immunoreactivity localized to engorged retinal tufts. Hypoxia upregulated VEGF, IGF-1, their receptors, and HIF-1α. Propranolol dose-dependently reduced upregulated VEGF and decreased hypoxic levels of IGF-1 mRNA and HIF-1α. Blockade of IGF-1R activity with PPP did not influence propranolols effects on VEGF. Retinal VEGF in normoxic mice or VEGF in brain, lungs, and heart of the OIR mice were unaffected by propranolol. Propranolol ameliorated the retinopathy score, restored occludin and albumin, and reduced hypoxia-induced plasma extravasation without influencing the vascular permeability induced by intravitreal VEGF. CONCLUSIONS This is the first demonstration that β-AR blockade is protective against retinal angiogenesis and ameliorates BRB dysfunction in OIR. Although the relevance of these results to infant ROP is uncertain, the findings may help to establish potential pharmacologic targets based on β3-AR pharmacology.

The metabotropic glutamate receptor 5 is necessary for late-phase long-term potentiation in the hippocampal CA1 region.

Selective antagonists of the metabotropic receptors 1 (mGluR1), +/-2-methyl-4-carboxyphenylglycine (LY367385), and mGluR5, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), were used to investigate the role of group I metabotropic receptors in late-phase long-term potentiation (L-LTP) at Schaffer collateral/commissural fiber-CA1 synapses in rat hippocampal slices. L-LTP was induced with three trains of tetanization of 1 s duration at 100 Hz separated by 10-min intervals. Neither LY367385 nor MPEP affected basal synaptic responses at the doses used (200 and 10 microM, respectively) and only the mGluR5 inhibitor MPEP blocked L-LTP. However, in agreement with previous mouse mutant studies, we found that both LY367385 and MPEP inhibited the induction of an LTP obtained with a single train of tetanization of 1 s duration at 100 Hz. MPEPs ability to disrupt L-LTP was not due to an effect on NMDA responses since it did not affect pharmacologically isolated N-methyl-D-aspartate (NMDA) excitatory postsynaptic potentials (EPSPs). However, MPEP prevented the increased phosphorylation in dendrites of p70 S6 kinase (p70(S6K)) at Thr3889, a major regulator of translation required for the induction of protein synthesis-dependent forms of LTP.

γ-Hydroxybutyrate inhibits excitatory postsynaptic potentials in rat hippocampal slices

Abstract γ-Hydroxybutyrate (GHB) has been shown to mimic different central actions of ethanol, to suppress alcohol withdrawal syndrome, and to reduce alcohol consumption both in rats and in humans. The aim of the present study was to determine if GHB shared with alcohol the ability to inhibit glutamate action at both NMDA and AMPA/kainate receptors. The NMDA or the AMPA/kainate receptors-mediated postsynaptic potentials were evoked in CA1 pyramidal neurons by stimulation of Schaffer-collateral commissural fibers in the presence of CGP 35348, bicuculline to block the GABA B and GABA A receptors, and 10 μM 6,7-dinitroquinoxaline-2,3-dione (DNQX) or 30 μM dl -2-amino-5-phosphonovalerate (d-APV) to block AMPA/kainate or NMDA receptors, respectively. GHB (600 μM) produced a depression of both NMDA and AMPA/kainate receptors-mediated excitatory postsynaptic potentials with recovery on washout. The GHB receptors antagonist, NCS-382, at the concentration of 500 μM had no effect per se on these responses but prevented the depressant effect of GHB (600 μM) on the NMDA and AMPA/kainate-mediated responses. In the paired-pulse experiments, GHB (600 μM) depressed the amplitude of the first and the second evoked AMPA/kainate excitatory postsynaptic potentials, and significantly increased the paired-pulse facilitation (PPF). These results suggest that GHB inhibits excitatory synaptic transmission at Schaffer-collateral commissural–pyramidal neurons synapses by decreasing the probability of release of glutamate.

Somatostatin receptors differentially affect spontaneous epileptiform activity in mouse hippocampal slices

Somatostatin‐14 [somatotropin release‐inhibiting factor (SRIF)] reduces hippocampal epileptiform activity but the contribution of its specific receptors (sst1−5) is poorly understood. We have focused on the role of sst1 and sst2 in mediating SRIF modulation of epilepsy using hippocampal slices of wild‐type (WT) and sst1 or sst2 knockout (KO) mice. Recordings of epileptiform discharge induced by Mg2+‐free medium with 4‐aminopyridine were performed from the CA3 region before and after the application of SRIF compounds. In WT mice, SRIF and the sst1 agonist CH‐275 reduce epilepsy whereas sst1 blockade with its antagonist SRA‐880 increases the bursting discharge. Activation of sst2 does not affect the bursting frequency unless its agonist octreotide is applied with SRA‐880, indicating that sst1 masks sst2‐mediated modulation of epilepsy. In sst1 KO mice: (i) the bursting frequency is lower than in WT; (ii) SRIF, CH‐275 and SRA‐880 are ineffective on epilepsy and (iii) octreotide is also devoid of effects, whereas blockade of sst2 with the antagonist d‐Tyr8 Cyn 154806 increases the bursting frequency. In sst2 KO mice, the SRIF ligand effects are similar to those in WT. In the whole hippocampus of sst1 KO mice, sst2 mRNA, protein and binding are higher than in WT and reverse transcription‐polymerase chain reaction of the CA3 subarea confirms an increase of the sst2 messenger. We conclude that sst1 mediates inhibitory actions of SRIF and that interactions between sst1 and sst2 may prevent sst2 modulation of epilepsy. We suggest that, in sst1 KO mice, activation of over‐expressed sst2 reduces the bursting frequency, indicating that sst2 density represents the rate‐limiting factor for sst2‐mediated modulation of epilepsy.

Compensatory changes in the hippocampus of somatostatin knockout mice: upregulation of somatostatin receptor 2 and its function in the control of bursting activity and synaptic transmission

Somatostatin‐14 (SRIF) co‐localizes with γ‐aminobutyric acid (GABA) in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of seizures has been proposed, although its exact contribution requires some clarification. In particular, SRIF knockout (KO) mice do not exhibit spontaneous seizures, indicating that compensatory changes may occur in KO. In the KO hippocampus, we examined whether specific SRIF receptors and/or the cognate peptide cortistatin‐14 (CST) compensate for the absence of SRIF. We found increased levels of both sst2 receptors (sst2) and CST, and we explored the functional consequences of sst2 compensation on bursting activity and synaptic responses in hippocampal slices. Bursting was decreased by SRIF in wild‐type (WT) mice, but it was not affected by either CST or sst2 agonist and antagonist. sst4 agonist increased bursting frequency in either WT or KO. In WT, but not in KO, its effects were blocked by agonizing or antagonizing sst2, suggesting that sst2 and sst4 are functionally coupled in the WT hippocampus. Bursting was reduced in KO as compared with WT and was increased upon application of sst2 antagonist, while SRIF, CST and sst2 agonist had no effect. At the synaptic level, we observed that in WT, SRIF decreased excitatory postsynaptic potentials which were, in contrast, increased by sst2 antagonist in KO. We conclude that sst2 compensates for SRIF absence and that its upregulation is responsible for reduced bursting and decreased excitatory transmission in KO mice. We suggest that a critical density of sst2 is needed to control hippocampal activity.

Mechanisms underlying somatostatin receptor 2 down-regulation of vascular endothelial growth factor expression in response to hypoxia in mouse retinal explants†

Hypoxia is a trigger of VEGF expression, the primary cause of retinal pathologies characterized by neovascularization. During hypoxia, transcription factors such as STAT3 and HIF‐1 promote the increase in VEGF expression. Octreotide, a somatostatin receptor 2 (sst2)‐preferring agonist, reduces retinal VEGF expression and neovascularization. To investigate the intracellular pathways linking sst2 activation to the inhibition of hypoxia‐induced VEGF up‐regulation, we used pharmacological approaches and siRNA in mouse retinal explants cultured in normoxia or hypoxia. In hypoxic explants in which STAT3 or HIF‐1 was inhibited, we observed the existence of reciprocal interactions between STAT3 and HIF‐1, which synergistically induced VEGF expression. Octreotide prevented hypoxia‐induced activation of STAT3 and HIF‐1, and the downstream increase in VEGF expression, as evaluated in hypoxic explants treated with pharmacological inhibitors of STAT3 or HIF‐1 and in normoxic explants in which pharmacological activators of STAT3 or HIF‐1 were used to mimic a hypoxia‐like response. The effect of octreotide on STAT3 activation is in part indirect, through the blockade of VEGFR‐2 phosphorylation. The effect of octreotide on STAT3, HIF‐1, VEGFR‐2, and VEGF required Src homology region 2 domain‐containing phosphatase 1 (SHP‐1). In hypoxic extracts, octreotide induced SHP‐1 phosphorylation and activation, and inhibiting SHP‐1 abolished the octreotide effect on STAT3, HIF‐1, VEGFR‐2, and VEGF. The central role of SHP‐1 in the modulation of STAT3 and HIF‐1 was confirmed in normoxic explants in which pharmacologically activated SHP‐1 prevented the effect of STAT3 or HIF‐1 activation. Immunohistochemical studies showed that under hypoxia sst2 and VEGF are expressed by retinal vessels, thus indicating a possible direct effect of octreotide on VEGF‐containing endothelial cells. These data clarify the mechanism by which octreotide prevents hypoxia‐induced VEGF up‐regulation and support the effectiveness of octreotide in treatment of oxygen‐induced retinopathies. These results may have implications in designing therapies targeting STAT3 and/or HIF‐1 aimed at preventing retinal neovascularization. Copyright

Gamma-hydroxybutyrate Reduces GABAA-mediated Inhibitory Postsynaptic Potentials in the CA1 Region of Hippocampus

Gamma-hydroxybutyric acid (GHB) is a psychoactive drug and a putative neurotransmitter, derived from gamma-aminobutyric acid (GABA). At micromolar concentrations GHB binds to specific high and low affinity binding sites present in discrete areas of the brain, while at millimolar concentrations GHB also binds to GABAB receptors. Previous studies indicated that GHB inhibits both NMDA and AMPA receptor mediated excitatory postsynaptic potentials in hippocampal CA1 pyramidal neurons. This action of GHB occurs in the presence of GABAB blockade and is antagonized by NCS-382, a specific GHB receptor antagonist, suggesting that it is mediated by GHB receptors. In the present study, we have investigated the effect of GHB on GABAA mediated inhibitory postsynaptic potentials (GABAA-IPSP) elicited in CA1 hippocampal pyramidal neurons by stimulation of Schaffer collateral-commissural fibers. We observed that GHB inhibited GABAA-IPSPs by about 40% at concentrations of 300–600 μM. GHB inhibition was blocked by NCS-382 (500 μM), which per se failed to modify GABAA-IPSPs. Moreover, GHB failed to modify cell membrane depolarization induced by the brief pressure application of GABA in the presence of tetrodotoxin (TTX), indicating that GHB does not inhibit postsynaptic GABA responses. However, GHB reduced the amplitude of GABAA-IPSPs elicited in pyramidal neurons by paired pulse stimulation and enhanced paired pulse facilitation with respect to control condition, suggesting that GHB reduces GABA release from nerve terminals. Finally, GHB failed to reduce the amplitude of GABAA-IPSPs in the presence of BaCl2, suggesting that the effect of GHB is due to GHB receptor-mediated presynaptic inhibition of Ca2+ influx.

Effects of Somatostatin Analogues on Retinal Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy: Involvement of the Somatostatin Receptor Subtype 2

PURPOSE To determine whether selective activation or blockade of the somatostatin (SRIF) receptor 2 (sst(2)) with two SRIF analogues, octreotide and D-Tyr(8) cyanamid 154806 (CYN), influences retinal vascularization and levels of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and -2 in a mouse model of oxygen-induced retinopathy (OIR). METHODS Wild-type (WT), sst(1)-knockout (KO), and sst(2)-KO mice were used. The OIR model was used to test the effects of octreotide and CYN administered subcutaneously. Retinopathy was assessed by a retinal scoring system using fluorescein-perfused retinal wholemounts. Retinal levels of VEGF, VEGFR-1, and -2 were evaluated with quantitative RT-PCR, Western blot, and ELISA. RESULTS In both WT and sst(1)-KO mice, OIR-induced neovascularization was reduced by octreotide, whereas it was increased by CYN. No effects of octreotide and CYN on retinal neovascularization were observed in sst(2)-KO retinas. Hypoxia upregulated the expression of VEGF and its receptors. Compared with WT retinas, the increase in VEGF, but not in VEGF receptors, was less pronounced in sst(1)-KO retinas in which sst(2) is known to be overexpressed. The hypoxia-induced increase in VEGF and its receptors was affected by SRIF analogues, with ameliorative effects of octreotide and worsening effects of CYN, which were more pronounced in the presence of sst(2) overexpression. CONCLUSIONS These data suggest that sst(2) regulates angiogenic responses to the hypoxic insult through a modulation of retinal levels of VEGF and its receptors. The present results further support the possibility of the use of sst(2)-selective ligands in the treatment of retinopathy.

Vascular endothelial growth factor up-regulation in the mouse hippocampus and its role in the control of epileptiform activity

The vascular endothelial growth factor (VEGF) signalling pathway may represent an endogenous anti‐convulsant in the rodent hippocampus although its exact contribution requires some clarification. In mouse hippocampal slices, the potassium channel blocker 4‐aminopyridine (4‐AP) in the absence of external Mg2+(0 Mg2+) produces both ictal and interictal activity followed by a prolonged period of repetitive interictal activity. In this model, we demonstrated that exogenous VEGF has clear effects on ictal and interictal activity as it reduces the duration of ictal‐like events, but decreases the frequency and intensity of interictal discharges. VEGF affects epileptiform activity through its receptor VEGFR‐2. We also demonstrated for the first time that the synaptic action of VEGF in the hippocampus is through VEGFR‐2‐mediated effects on NMDA and GABAB receptors and that VEGF does not affect the NMDA excytatory postsynaptic potential paired‐pulse facilitation ratio. Exogenous VEGF does not affect the AMPA‐mediated responses and the dendritic or the somatic GABAA inhibitory postsynaptic potentials. In addition, VEGF drastically reduces 0 Mg2+/4‐AP‐induced glutamate release through VEGFR‐2 activation. In vitro epileptiform activity is sufficient to increase hippocampal expression of VEGF and VEGFR‐2, and this up‐regulation may serve a neuroprotective and/or anti‐convulsant role. VEGFR‐2 up‐regulation has been localized to the CA1 region, which suggests that VEGF signalling may protect CA1 pyramidal cells from hyperexcitability. These results indicate that VEGF controls epileptic activity by influencing both glutamatergic and GABAergic transmission and further advance our understanding of the conditions required for endogenous VEGF up‐regulation, and the mechanisms by which VEGF achieves an anti‐convulsant effect.