Donata Rodi

Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor–2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures.

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.

Inhibitory effect of nociceptin on [3H]-5-HT release from rat cerebral cortex slices

The effect of nociceptin (NC) on 5‐hydroxytryptamine (5‐HT) release was studied in rat cerebral cortex slices preincubated with [3H]‐5‐HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion. NC (0.1–3 μM), present in the medium from the 70th min onward, concentration‐dependently reduced electrically evoked [3H]‐5‐HT efflux (pEC50=6.54, Emax −54%). The inhibition was not antagonized by naloxone (1 μM) ruling out the involvement of opioid receptors. Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2, which acts as an opioid‐like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]‐5‐HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1ψ(CH2‐NH2)Gly2]NC(1‐13)NH2 prevented the action of NC added at the 70th min. The non‐selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 μM), in the presence of 10 μM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect. These findings demonstrate that NC inhibits 5‐HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5‐HT.

Protein kinase C activity, translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia

Protein kinase C (PKC) involvement in ischemia‐induced neuronal damage has been investigated in superfused rat cerebral cortex slices submitted to 15 min of oxygen‐glucose deprivation (OGD) and in primary cultures of rat cortical neurons exposed to 100 μM glutamate (GLU) for 10 min. OGD significantly increased the total PKC activity in the slices, mostly translocated in the particulate fraction. After 1 hr of reperfusion, the total PKC activity was reduced and the translocated fraction dropped by 84% with respect to the control. Western blot analysis of OGD samples showed an increase in total β2 and ϵ PKC isoform levels. After reperfusion, the total levels of α, β1, β2 and γ isoforms were significantly reduced, whereas the ϵ isoform remained at an increased level. Endogenous GLU release from OGD slices increased to about 15 times the basal values after 15 min of oxygen‐glucose deprivation, and to 25 and 35 times the basal level in the presence of the PKC inhibitors staurosporine (0.1 μM) and bisindolylmaleimide (1 μM), respectively. Western blot analysis of GLU‐treated cortical neurons showed a significant decrease only in the total level of β2 isoforms. Cell survival was reduced to 31% in GLU‐treated neuronal cultures; PKC inhibitors were not able to modify this effect. These findings demonstrate that the cell response to OGD and GLU involves PKC in a complex way. The net role played by PKC during OGD may be to reduce GLU release and, consequently, neurotoxicity. The isoforms β2 and ϵ are affected the most and may play a significant role in the mechanisms underlying neurotoxicity/neuroprotection.

FGF-2 Overexpression Increases Excitability and Seizure Susceptibility but Decreases Seizure-Induced Cell Loss

Fibroblast growth factor 2 (FGF-2) has multiple, pleiotropic effects on the nervous system that include neurogenesis, neuroprotection and neuroplasticity. Thus, alteration in FGF-2 expression patterns may have a profound impact in brain function, both in normal physiology and in pathology. Here, we used FGF-2 transgenic mice (TgFGF2) to study the effects of endogenous FGF-2 overexpression on susceptibility to seizures and to the pathological consequences of seizures. TgFGF2 mice display increased FGF-2 expression in hippocampal pyramidal neurons and dentate granule cells. Increased density of glutamatergic synaptic vesicles was observed in the hippocampus of TgFGF2 mice, and electrophysiological data (input/output curves and patch-clamp recordings in CA1) confirmed an increase in excitatory inputs in CA1, suggesting the presence of a latent hyperexcitability. Indeed, TgFGF2 mice displayed increased susceptibility to kainate-induced seizures compared with wild-type (WT) littermates, in that latency to generalized seizure onset was reduced, whereas behavioral seizure scores and lethality were increased. Finally, WT and TgFGF2 mice with similar seizure scores were used for examining seizure-induced cellular consequences. Neurogenesis and mossy fiber sprouting were not significantly different between the two groups. In contrast, cell damage (assessed with Fluoro-Jade B, silver impregnation and anti-caspase 3 immunohistochemistry) was significantly lower in TgFGF2 mice, especially in the areas of overexpression (CA1 and CA3), indicating reduction of seizure-induced necrosis and apoptosis. These data suggest that FGF-2 may be implicated in seizure susceptibility and in seizure-induced plasticity, exerting different, and apparently contrasting effects: favoring ictogenesis but reducing seizure-induced cell death.

Pro-nociceptin/orphanin FQ and NOP receptor mRNA levels in the forebrain of food deprived rats.

Forebrain injections of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP opioid receptor, previously referred to as ORL1 or OP4 receptor, stimulate feeding in freely feeding rats, while the NOP receptor antagonist [Nphe(1)]N/OFQ(1-13)NH(2) inhibits food deprivation-induced feeding. To further evaluate whether the N/OFQ-NOP receptor system plays a physiological role in feeding control, the present study evaluated forebrain mRNA levels for the N/OFQ precursor (pro-N/OFQ), as well as for the NOP receptor in food deprived rats. The results obtained show that food deprived rats have lower mRNA levels for the NOP receptor in several forebrain regions; a significant reduction was found in the paraventricular and lateral hypothalamic nuclei and in the central nucleus of the amygdala. Food deprived rats also exhibited lower pro-N/OFQ mRNA levels in the central amygdala. These results suggest that the N/OFQ-NOP receptor system may have a physiological role in feeding control. The observation that food deprivation reduces gene expression of the N/OFQ-NOP receptor system is apparently not consistent with a direct hyperphagic action for N/OFQ. Taking into account that N/OFQ exerts inhibitory actions at cellular level, the present results may be in keeping with the hypothesis that N/OFQ stimulates feeding by inhibiting neurons inhibitory for food intake; under conditions of food deprivation, these neurons may be silent and the N/OFQ-NOP receptor system, which controls them, may also be regulated at a lower level. Consistently, in the present study N/OFQ stimulated food intake in freely feeding rats, but did not further increase feeding in food deprived rats.

Expression of kinin B1 receptors in the spinal cord of streptozotocin-diabetic rat.

Previous studies have reported cardiovascular and nociceptive responses after intrathecal injection of kinin B1 receptor (B1R) agonists in the model of streptozotocin (STZ)-diabetic rat (diabetic). The aim of this study was to measure the early up-regulation of B1R binding sites and mRNA in the thoracic spinal cord of diabetic and control rats. Data show significant increases of specific B1R binding sites in the dorsal horn of diabetic rats 2 days (+315%), 7 days (+303%) and 21 days (+181%) after STZ treatment. Levels of mRNA were significantly increased (+68%) at 2 and 7 days but not at 21 days. These data bring the first molecular evidence for an early up-regulation of B1R in the spinal cord of diabetic rat.

Induction of B1 bradykinin receptors in the kindled hippocampus increases extracellular glutamate levels: A microdialysis study

A link between temporal lobe epilepsy (the most common epileptic syndrome in adults) and neuropeptides has been established. Among neuropeptides, the possible involvement of bradykinin has recently received attention. An autoradiographic analysis has shown that B1 receptors, which are physiologically absent, are expressed at high levels in the rat brain after completion of kindling, a model of temporal lobe epilepsy. Thus, the present work aimed at investigating the functional implications of this observation, by studying the effect of B1 receptor activation on extracellular glutamate levels in the kindled hippocampus. Microdialysis experiments have been performed in two groups of rats, control and kindled. Glutamate outflow has been measured under basal conditions and after chemical stimulation with high K+ (100 mM in the dialysis solution). Basal glutamate outflow in kindled animals was significantly higher than in controls. High K+-evoked glutamate outflow was also more pronounced in kindled animals, consistent with the latent hyperexcitability of the epileptic tissue. The B1 receptor agonist Lys-des-Arg9-BK induced an increase of basal and high K+-evoked glutamate outflow in kindled but not in control rats, and the selective B1 receptor antagonist R-715 prevented both these effects. Furthermore, R-715 significantly reduced high K+-evoked glutamate outflow when applied alone. These data suggest that the bradykinin system contributes to the modulation of epileptic neuronal excitability through B1 receptors.

Effects of cholecystokinin tetrapeptide (CCK4) and of anxiolytic drugs on GABA outflow from the cerebral cortex of freely moving rats

The effect of cholecystokinin tetrapeptide (CCK(4)) and of different anxiolytic drugs on GABA outflow from the cerebral cortex was investigated in freely moving rats, by using the epidural cup technique. CCK(4) (3-30 microg/kg, i.p.) increased GABA outflow and induced objective signs of anxiety. These neurochemical and behavioral responses were prevented by the CCK(B) antagonist GV150013 at 0.1 microg/kg (i.p.). At higher doses (up to 30 microg/kg) this compound per se reduced GABA release and caused sedation, suggesting the presence of a CCKergic positive tonic modulation on GABA interneurons. Similarly the GABA(A) receptors modulator, diazepam (2mg/kg, i.p.) and the 5-HT(1A) agonist buspirone (3mg/kg, i.p.) reduced GABA outflow and caused the expected behavioral effects (reduced muscle tone, mild 5-HT syndrome) which were prevented by the respective, selective antagonists, flumazenil (1mg/kg, i.p.) and NAN-190 (3mg/kg, i.p.). These findings support the idea that GV150013, diazepam and buspirone inhibit GABAergic cortical activity, through the respective receptors. This neurochemical effect may represent the end-effect of various anxiolytic compounds affecting the cortical circuitry.

Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro

The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied. Rat, mouse and guinea‐pig cortical slices and synaptosomes were preincubated with 0.1 μM [3H]‐NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes. In rat cortical slices, 0.01–3 μM NC reduced the evoked [3H]‐NE efflux (Emax−54%), with a bell‐shaped concentration‐response curve, which regained its monotonic nature in the presence of either 0.1 μM naloxone (NX) or 30 μM bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 μM concentration. In the rat, both 1 μM [Phe1ψ(CH2‐NH)Gly2]NC(1‐13)NH2 and 10 μM [Nphe1]NC(1‐13)NH2 (NPhe) antagonised NC‐induced inhibition, without per se modifying [3H]‐NE efflux. The effects of 0.3–1 μM NC concentrations were partially prevented by 1 μM NX; 1 μM D‐Phe‐Cys‐Thr‐D‐Trp‐Orn‐Thr‐Pen‐Thr‐NH2 (CTOP) was also an effective antagonist, but 0.1 μM norbinaltorphimine was not. In the mouse cerebral cortex, NC‐induced inhibition of NE release (pEC50 6.87, Emax−61%, in the slices) was prevented by Nphe but was NX‐insensitive. In guinea‐pig cortical slices, NC effect (pEC50 6.22, Emax−38%) was prevented by Nphe, but was NX‐insensitive. These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL1 receptors. In the rat, μ opioid and GABAA receptors are involved as well.