Sergio Ferri

Nitric oxide down-regulates brain-derived neurotrophic factor secretion in cultured hippocampal neurons

The regulation of neurotrophin (NT) secretion is critical for many aspects of NT-mediated neuronal plasticity. Neurons release NTs by activity-regulated secretion pathways, initiated either by neurotransmitters and/or by existing NTs by a positive-feedback mechanism. This process depends on calcium release from intracellular stores. Little is known, however, about potential pathways that down-regulate NT secretion. Here we demonstrate that nitric oxide (NO) induces a rapid down-regulation of brain-derived neurotrophic factor (BDNF) secretion in cultured hippocampal neurons. Similar effects occur by activating a downstream target of intracellular NO, the soluble guanylyl cyclase, or by increasing the levels of its product, cGMP. Furthermore, down-regulation of BDNF secretion is mediated by cGMP-activated protein kinase G, which prevents calcium release from inositol 1,4,5-trisphosphate-sensitive stores. Our data indicate that the NO/cGMP/protein kinase G pathway represents a signaling mechanism by which neurons can rapidly down-regulate BDNF secretion and suggest that, in hippocampal neurons, NT secretion is finely tuned by both stimulatory and inhibitory signals.

Chronic opiate agonists down-regulate prodynorphin gene expression in rat brain

The effects of long-term administration of opioid agonists on the regulation of prodynorphin gene expression in rat brain were investigated. Chronic intracerebroventricular treatment with the synthetic opioid agonist acting on the kappa receptor, U-50,488H, and the classic mu agonist morphine markedly decreased prodynorphin mRNA levels in hypothalamus, hippocampus and striatum of tolerant rats. Levels of ir-Dynorphin A remained unchanged except in two cases. Chronic exposure to opiates thus appears to induce modifications of the endogenous opioid system, as regards gene expression regulation.

Distinguishable effects of intrathecal dynorphins, somatostatin, neurotensin and s-calcitonin on nociception and motor function in the rat

&NA; We determined the effects on nociceptive threshold and motor function of dynorphin‐gene products, dynorphin A‐(1–32) (DYN A‐(1–32), DYN A‐(1–8), DYN B and DYN B‐29 and the non‐opioid peptides somatostatin, neurotensin and salmon calcitonin (s‐CT) after intrathecal administration in the rat. DYN A‐(1–32) (25 nmol) produced maximal elevation of tail‐flick latency accompanied by severe hind limb paralysis and tail flaccidity lasting 6 h and still present at 24 h in several animals. Antinociception evaluated by the vocalization test wore off within 2 h. A lower dose of the peptide (6.25 nmol) did not alter the tail‐flick reflex and motor function but significantly elevated the vocalization threshold. The other dynorphins showed weaker, short‐lasting activity on the nociceptive threshold, the order of potency being as follows: DYN B‐29 > DYN B > DYN A‐(1–8). On the other hand, at the high doses DYN B (100 nmol) and DYN B‐29 (50 and 100 nmol) caused moderately severe hind limb paralysis whereas DYN A‐(1–8) did not cause any motor impairment up to the dose of 100 nmol. MR 1452, a relatively preferential antagonist of the &kgr; opioid receptor, prevented both the antinociceptive and motor effects of dynorphins. Intrathecal somatostatin (25 nmol) had a profile of activity superimposable on that of DYN A‐(1–32): long‐lasting (up to 24 h) elevation of tail‐flick latency with hind limb paralysis and a shorter (4 h) elevation of the vocalization threshold. MR 1452 did not modify these effects. Intrathecal neurotensin (25 nmol) and s‐CT (0.5 nmol) did not alter tail‐flick latency or vocalization threshold. However, adopting the hot plate as the analgesimetric test, both peptides elevated the time of hind paw licking, taken as an index of nociception. No signs of motor dysfunction were observed at the doses employed.

Central and peripheral sites of action for the protective effect of opioids of the rat stomach.

Rats exposed to combined cold and restraint exhibited a reduced intensity of gastric damage when pre-treated intraperitoneally with morphine HCl or with the synthetic enkephalin analog [D-Ala2, MePhe4, Met(0)5ol]enkephalin (FK 33-824). Morphine HCl and FK 33-824 prevented some of the indices of the lesion also when injected intracerebroventricularly; morphine methyliodide, quaternary derivative of morphine with does not cross the blood brain barrier, was fully effective, by intraperitoneal route, in preventing the gastric damage. Both peripheral and central mechanisms seem, therefore, involved in the protective effect of opioids on rat gastric mucosa. Morphine HCl and FK 33-824 reduced significantly gastric acid secretion when administered intracerebroventricularly or intraperitoneally; in addition, a concomitant increase of prostaglandin production was observed in rat gastric mucosa after i.p. administration of both opioids. Both these events might contribute to the protective action of opioids on the stomach.

Antinociceptive profile of intracerebroventricular salmon calcitonin and calcitonin gene-related peptide in the mouse formalin test

The effects of intracerebroventricularly administered salmon calcitonin (sCT) and calcitonin gene-related peptide (CGRP) on the behavioural response of the mouse to formalin injections were investigated. Mice lick their hindpaws for 5 min after formalin injections, then stop, and resume intensive licking for another 10 min beginning 20 min after the injections. Both peptides reduced the nociceptive response in the two phases of the test (0-5 and 20-30 min after formalin injection). Antinociceptive A50 values were 3.3 micrograms/mouse and 4.7 micrograms/mouse respectively in the first phase for sCT and CGRP. The effects of sCT and CGRP appeared to be dose-dependent in the first phase. Since in the second phase sCT appeared more effective and CGRP gave a bell-shaped curve, possible differences in the mechanisms of action of the peptides in the two response intervals of the test are suggested.

Antinociceptive activity of salmon calcitonin injected intrathecally in the rat

Salmon calcitonin injected intrathecally in unanesthetized rats produced long-lasting, dose-dependent elevations of nociceptive threshold as measured in the hot plate test. This antinociceptive action was nonopiate in nature as it was uninfluenced by the narcotic antagonists naloxone and MR 1452; moreover, the peptide was still able to raise the nociceptive threshold in morphine-tolerant rats. It is suggested that the spinal cord may represent one of the sites of action for calcitonin-induced antinociception.

Chronic intracerebroventricular cocaine differentially affects prodynorphin gene expression in rat hypothalamus and caudate-putamen

We investigated the effects of sustained administration of cocaine on the regulation of prodynorphin gene expression in rat brain. Intracerebroventricular (i.c.v.) infusion of cocaine hydrochloride (30 micrograms/day) for 7 days, by means of osmotic minipumps, elicited a significant 35% decrease of prodynorphin mRNA levels in rat hypothalamus and increase (22%) in caudate-putamen. At the same time and in the same animals, no significant changes were detected in the hippocampus or in the nucleus accumbens. These results indicate that continuously infused cocaine is able to modulate expression of the prodynorphin gene in opposite directions or has no effect on prodynorphin expression, depending on the brain region analysed. Cocaine, as well as opiates, might activate specific neuronal pathways, shared by different classes of drugs of abuse, involving, at least in part, the endogenous opioid system.

Effect of ω-conotoxin and verapamil on antinociceptive, behavioural and thermoregulatory responses to opioids in the rat

This study with the rat evaluated the contribution of omega-conotoxin GVIA-(omega-CgTx) and verapamil-sensitive Ca2+ channels in behavioural, antinociceptive and thermoregulatory responses to intracerebroventricular (i.c.v.) injection of [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and dynorphin A-(1-17), which are selective agonists for putative mu, delta and kappa-opioid receptors, respectively. The rats treated with omega-CgTx (8-32 pmol i.c.v.) showed transient, dose-dependent shaking behaviour, hyperalgesia and hypothermia which gradually disappeared within 4 h. The behaviour of the rats was normal by 24 h. Histological examination of brain sections showed morphological alterations of neurons in the hippocampus, medial-basal hypothalamus and pyriform cortex. antinociception, catalepsy and thermoregulatory responses elicited by DAMGO (0.4 and 2.0 nmol) were significantly prolonged and potentiated by verapamil (20 pmol i.c.v. 15 min before) or omega-CgTx (8 pmol 24 h before). Antinociception and hypothermia induced by DPDPE were antagonized by verapamil and omega-CgTx, whereas only omega-CgTx prevented the behavioural arousal observed after DPDPE. Similarly, hypothermia induced by dynorphin A-(1-17) (5.0 nmol) and by the kappa-opioid receptor agonist U50,488H (215 nmol) was antagonized by the two Ca2+ channel blockers but only omega-CgTx prevented the barrel rolling and bizarre postures caused by the opioid peptide.

Supraspinal and spinal effects of [Phe1Ψ(CH2-NH)Gly2]-nociceptin(1–13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.

Early Changes in Prodynorphin mRNA and ir-Dynorphin A Levels after Kindled Seizures in the Rat

Prodynorphin mRNA and immunoreactive dynorphin A (ir‐dynorphin A) levels were measured in different brain areas at various time points after amygdala kindled seizures. In the hippocampus, striatum and hypothalamus, prodynorphin mRNA levels were not significantly changed in kindled rats (killed 1 week after the last stimulus‐evoked seizure), but they were significantly increased 1 h after seizures. The relative increase was the highest in the hippocampus (∼3‐fold). In the brainstem, midbrain and cerebral cortex no changes in prodynorphin mRNA were detected in kindled rats, 1 h or 1 week after a kindled seizure. ir‐Dynorphin A levels were significantly reduced in the hippocampus and in the striatum of kindled rats, as well as 5 and 60 min after kindled seizures, but they were increased back to control levels after 120 min. In the hypothalamus, ir‐dynorphin A levels were significantly increased 120 min after a kindled seizure. ir‐Dynorphin A levels were also significantly reduced in the brainstem and in the frontal, parietal and temporal cortex 120 min, but not 5 or 60 min, after a kindled seizure. Taken together, these data support the hypothesis that the dynorphinergic system is activated after amygdala kindled seizures, with different kinetics in different brain areas.