Nicoletta Galeotti

Local anaesthetic activity of β-caryophyllene ☆

Abstract In this work we studied the local anaesthetic activity of β-caryophyllene, one of the main components of clove oil obtained from the dried flower-buds of Syzygium aromaticum (Myrtaceae family). We compared its activity to a chemically related compound, caryophyllene oxide. Anaesthetic activity was evaluated in vivo in the rabbit conjunctival reflex test and in vitro in a rat phrenic nerve-hemidiaphragm preparation. β-Caryophyllene (10 −4 –1 μg/ml), but not caryophyllene oxide, was able to reduce drastically, in a dose-dependent manner, the electrically evoked contractions of the rat phrenic hemidiaphragm. In the rabbit, conjunctival reflex test treatment with a solution of β-caryophyllene (10–1000 μg/ml) allowed a dose-dependent increase in the number of stimuli necessary to provoke the reflex. As in the in vitro results, caryophyllene oxide was ineffective also in the in vivo test. In conclusion, these data evidence the local anaesthetic activity of β-caryophyllene, which appears to be strictly dependent on its chemical structure.

Signaling pathway of morphine induced acute thermal hyperalgesia in mice

Abstract Systemic administration of morphine induced a hyperalgesic response in the hot plate test, at an extremely low dose (1–10 μg/kg). We have examined in vivo whether morphine, at an extremely low dose, induces acute central hypernociception following activation of the opioid receptor‐mediated PLC/PKC inositol‐lipid signaling pathway. The PLC inhibitor U73122 and the PKC blocker, calphostin C, dose dependently prevented the thermal hypernociception induced by morphine. This effect was also prevented by pretreatment with aODN against PLCβ3 at 2 nmol/mouse and PKCγ at 2–3 nmol/mouse. Low dose morphine hyperalgesia was dose dependently reversed by selective NMDA antagonist MK801 and ketamine. This study demonstrates the presence of a nociceptive PLCβ3/PKCγ/NMDA pathway stimulated by low concentrations of morphine, through μOR1 receptor, in mouse brain. This signaling pathway appears to play an opposing role in morphine analgesia. When mice were treated with a morphine analgesic dose (7 mg/kg), the downregulation of PLCβ3 or PKCγ at the same aODN doses used for the prevention of the hyperalgesic effect induced, respectively, a 46% and 67% potentiation in analgesic response. Experimental and clinical studies suggest that opioid may activate pronociceptive systems, leading to pain hypersensitivity and short‐term tolerance, a phenomenon encountered in postoperative pain management by acute opioid administration. The clinical management of pain by morphine may be revisited in light of the identification of the signaling molecules of the hyperalgesic pathway.

Loss of muscarinic antinociception by antisense inhibition of M1 receptors

The effect on cholinergic analgesia of inactivation of the M1 gene by an antisense oligodeoxyribonucleotide (aODN) was investigated in the mouse hot plate test. Mice received a single intracerebroventricular (i.c.v.) injection of anti‐M1 aODN (0.3, 1.0 or 2.0 nmol per injection), degenerate ODN (dODN) or vehicle on days 1, 4 and 7. A dose‐dependent inhibition of the antinociception induced by the muscarinic agonists oxotremorine (0.1 mg kg−1 s.c.) and McN‐A‐343 (30 μg per mouse i.c.v.) and the cholinesterase inhibitor physostigmine (0.2 mg kg−1 s.c.) was observed 24 h after the last i.c.v. injection of aODN. Time‐course experiments revealed that, after the end of the aODN treatment, sensitivity to analgesic drugs progressively appeared reaching the normal range at 96 h. The anti‐M1 aODN was selective against muscarinic antinociception since the enhancement of pain threshold produced by morphine and baclofen were not affected by the above‐mentioned treatment. dODN, used as control, did not affect muscarinic antinociception. Binding studies evidenced a selective reduction of M1 receptor levels in the hippocampus of aODN‐treated mice. Neither aODN, dODN nor vehicle produced any behavioural impairment of mice as revealed by the rota‐rod and Animex experiments. These results indicate that activation of M1 muscarinic receptor subtype is fundamental to induce central cholinergic analgesia in mice.

Caffeine induces central cholinergic analgesia.

Abstract The antinociceptive effect of caffeine was examined by using the hot-plate, abdominal constriction tests in mice and the tail flick and paw-pressure tests in rats. Caffeine (1–5 mg kg–1 s.c. in mice; 2.5–5 mg kg–1 i.p. in rats) produced significant antinociception in both species which was prevented by atropine (5 mg kg–1 i.p.), pirenzepine (0.1 μg per mouse i.c.v.), hemicholinium-3 (1 μg/ mouse i.c.v.) and N6-cyclopentyladenosine (5 μg/mouse i.c.v.), but not by naloxone (1 mg kg–1 i.p.), CGP 35348 (100 mg kg–1 i.p.), α-methyl p-tyrosine (100 mg kg–1 i.p.) and reserpine (2 mg kg–1 i.p.). Intracerebroventricular injection of caffeine in mice at doses (2.5–5 μg per mouse) which were largely ineffective by parenteral routes, induces an antinociception whose intensity equalled that obtainable s.c. or i.p. In the antinociceptive dose-range, caffeine did not produce any behavioural impairment as revealed by the rotarod and Irwing tests. On the basis of the above data, it can be postulated that caffeine exerts an antinociceptive effect mediated by central amplification of cholinergic transmission.

Effect of potassium channel modulators in mouse forced swimming test

The effect of intracerebroventricular (i.c.v.) administration of different potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone), potassium channel openers (pinacidil, minoxidil, cromakalim) and aODN to mKv1.1 on immobility time was evaluated in the mouse forced swimming test, an animal model of depression. Tetraethylammonium (TEA; 5 μg per mouse i.c.v.), apamin (3 ng per mouse i.c.v.), charybdotoxin (1 μg per mouse i.c.v.) and gliquidone (6 μg per mouse i.c.v.) administered 20 min before the test produced anti‐immobility comparable to that induced by the tricyclic antidepressants amitriptyline (15 mg kg−1 s.c.) and imipramine (30 mg kg−1 s.c.). By contrast pinacidil (10–20 μg per mouse i.c.v.), minoxidil (10–20 μg per mouse i.c.v.) and cromakalim (20–30 μg per mouse i.c.v.) increased immobility time when administered in the same experimental conditions. Repeated administration of an antisense oligonucleotide (aODN) to the mKv1.1 gene (1 and 3 nmol per single i.c.v. injection) produced a dose‐dependent increase in immobility time of mice 72 h after the last injection. At day 7, the increasing effect produced by aODN disappeared. A degenerate mKv1.1 oligonucleotide (dODN), used as control, did not produce any effect in comparison with saline‐ and vector‐treated mice. At the highest effective dose, potassium channels modulators and the mKv1.1 aODN did not impair motor coordination, as revealed by the rota rod test, nor did they modify spontaneous motility as revealed by the Animex apparatus. These results suggest that modulation of potassium channels plays an important role in the regulation of immobility time in the mouse forced swimming test.

Involvement of potassium channels in amitriptyline and clomipramine analgesia

The effect of the administration of modulators of different subtypes of K(+) channels on antinociception induced by the tricyclic antidepressants amitriptyline and clomipramine was evaluated in the mouse hot plate test. The administration of the voltage-gated K(+) channel blocker tetraethylammonium (0.01-0.5 microg per mouse i.c.v. ) prevented antinociception induced by both amitriptyline (15 mg kg(-1) s.c.) and clomipramine (25 mg kg(-1) s.c.). The K(ATP) channel blocker gliquidone (0.1-1.0 microg per mouse i.c.v.) prevented antinociception produced by amitriptyline and clomipramine whereas the K(ATP) channel openers minoxidil (10 microg per mouse i. c.v.) and pinacidil (25 microg per mouse i.c.v.) potentiated tricyclic antidepressant-induced analgesia. The administration of the Ca(2+)-gated K(+) channel blocker apamin (0.1-1.0 ng per mouse i. c.v.) completely prevented amitriptyline and clomipramine analgesia. At the highest effective doses, none of the drugs used induced behavioural side effects or impaired motor coordination, as revealed by the rota-rod test, spontaneous motility or inspection activity, as revealed by the hole board test. The present results demonstrate that central antinociception induced by amitriptyline and clomipramine involves the opening of different subtypes of K(+) channels (voltage-gated, K(ATP) and Ca(2+)-gated) which, therefore, represent a step in the transduction mechanism of tricyclic antidepressant analgesia.

The phospholipase C-IP3 pathway is involved in muscarinic antinociception.

The cellular events involved in muscarinic analgesia were investigated in the mouse hot-plate test. Intracerebroventricular (i.c.v.) pretreatment with antisense oligonucleotides (aODNs) against the α subunit of Gq and G11 proteins prevented the analgesia induced by physostigmine and oxotremorine. Furthermore, administration of the phospholipase C (PLC) inhibitor U-73122, as well as the injection of an aODN complementary to the sequence of PLCβ1, antagonized the increase of the pain threshold induced by both cholinomimetic drugs. In mice undergoing treatment with LiCl, which impairs phosphatidylinositol synthesis, or treatment with heparin, an IP3 receptor antagonist, the antinociception induced by physostigmine and oxotremorine was dose-dependently antagonized. I.c.v. pretreatment with TMB-8, a blocker of Ca2+ release from intracellular stores, prevented the increase of pain threshold induced by the investigated cholinomimetic drugs. Coadministration of Ca2+ restored the muscarinic analgesia in LiCl, heparin, and TMB-8-preatreated mice. On the other hand, i.c.v. pretreatment with the selective protein kinase C (PKC) inhibitor calphostin C, resulted in a dose-dependent enhancement of physostigmine- and oxotremorine-induced antinociception. The administration of PKC activators, such as PMA and PDBu, dose dependently prevented the cholinomimetic drug-induced increase of pain threshold. Neither aODNs nor pharmacological treatments employed produced any behavioral impairment of mice as revealed by the rota-rod and hole-board tests. These results indicate a role for the PLC-IP3 pathway in central muscarinic analgesia in mice. Furthermore, activation of PKC by cholinomimetic drugs may represent a pathway of negative modulation of muscarinic antinociception.

Satiety factor oleoylethanolamide recruits the brain histaminergic system to inhibit food intake

Significance Several endogenous molecules contribute to the building of a complex network of neural and hormonal signals that align food intake and energy expenditure. Cerebral histamine works as a satiety factor by activating histamine H1 receptor (H1R) in specific hypothalamic nuclei. Indeed, atypical antipsychotics presumably cause obesity by targeting brain H1R. The endogenous lipid messenger oleoylethanolamide (OEA) mediates fat-induced satiety by engaging sensory fibers of the vagus nerve that project centrally. We find that depletion of brain histamine blunts OEA-induced hypophagia in mice. Our study uncovers previously unidentified neural signaling mechanisms involved in the anorectic action of OEA. Our data offer new perspectives for developing more effective and safer pharmacotherapies to treat obesity and ameliorate the profile of centrally acting drugs. Key factors driving eating behavior are hunger and satiety, which are controlled by a complex interplay of central neurotransmitter systems and peripheral stimuli. The lipid-derived messenger oleoylethanolamide (OEA) is released by enterocytes in response to fat intake and indirectly signals satiety to hypothalamic nuclei. Brain histamine is released during the appetitive phase to provide a high level of arousal in anticipation of feeding, and mediates satiety. However, despite the possible functional overlap of satiety signals, it is not known whether histamine participates in OEA-induced hypophagia. Using different experimental settings and diets, we report that the anorexiant effect of OEA is significantly attenuated in mice deficient in the histamine-synthesizing enzyme histidine decarboxylase (HDC-KO) or acutely depleted of histamine via interocerebroventricular infusion of the HDC blocker α-fluoromethylhistidine (α-FMH). α-FMH abolished OEA-induced early occurrence of satiety onset while increasing histamine release in the CNS with an H3 receptor antagonist-increased hypophagia. OEA augmented histamine release in the cortex of fasted mice within a time window compatible to its anorexic effects. OEA also increased c-Fos expression in the oxytocin neurons of the paraventricular nuclei of WT but not HDC-KO mice. The density of c-Fos immunoreactive neurons in other brain regions that receive histaminergic innervation and participate in the expression of feeding behavior was comparable in OEA-treated WT and HDC-KO mice. Our results demonstrate that OEA requires the integrity of the brain histamine system to fully exert its hypophagic effect and that the oxytocin neuron-rich nuclei are the likely hypothalamic area where brain histamine influences the central effects of OEA.

Influence of potassium channel modulators on cognitive processes in mice

1 The effect of i.c.v. administration of different potassium channel openers (minoxidil, pinacidil, cromakalim) and potassium channel blockers (tetraethylammonium, apamin, charybdotoxin, gliquidone, glibenclamide) on memory processes was evaluated in the mouse passive avoidance test. 2 The administration of minoxidil (10 μg per mouse i.c.v.), pinacidil (5–25 μg per mouse i.c.v.) and cromakalim (10–25 μg per mouse i.c.v.) immediately after the training session produced an amnesic effect. 3 Tetraethylammonium (TEA; 1–5 μg per mouse i.c.v.), apamin (10 ng per mouse i.c.v.), charybdotoxin (1 μg per mouse i.c.v.), gliquidone (3 μg per mouse i.c.v.) and glibenclamide (1 μg per mouse i.c.v.), administered 20 min before the training session, prevented the potassium channel opener‐induced amnesia. 4 At the highest effective doses, none of the drugs impaired motor coordination, as revealed by the rota rod test, or modified spontaneous motility and inspection activity, as revealed by the hole board test. 5 These results suggest that the modulation of potassium channels plays an important role in the regulation of memory processes. On this basis, the potassium channel blockers could be useful in the treatment of cognitive deficits.

Alpha-2 agonist-induced memory impairment is mediated by the alpha-2A-adrenoceptor subtype

The activation of alpha2-adrenoceptors has been reported to impair memory functions in both rats and humans. The alpha2-adrenoceptor subtype responsible for this detrimental effect is still unknown. The effect of the alpha2-agonists clonidine and guanabenz on memory processes, in dependence to the time of administration, was evaluated in the mouse passive avoidance test. Clonidine (0.02-0.2 mg kg(-1) i.p.) and guanabenz (0.1-0.3 mg kg(-1) i.p.) induced amnesia in a dose-dependent manner. From time-course experiments emerged that the impairment of memory function was detectable only when clonidine and guanabenz were administered 60 min before or immediately after the training test, respectively. This detrimental effect was prevented by pretreatment with the alpha2-antagonist yohimbine (1-3 mg kg(-1) i.p.) and by the alpha2A-antagonist BRL-44408 (0.3-1 mg kg(-1) i.p.). By contrast, the alpha(2B,C) antagonists ARC-239 (10 mg kg(-1) i.p.) and prazosin (1 mg kg(-1) i.p.) did not revert the amnesia induced by both clonidine and guanabenz. At the highest effective doses, clonidine and guanabenz were devoid of behavioral side-effects as well as maintained unaltered the motor coordination, as revealed by the rota-rod test. Furthermore, none of the compounds used modified the spontaneous motility as indicated by the Animex apparatus. These results indicate that clonidine and guanabenz impaired memory processes in a mouse passive avoidance paradigm through the selective activation of the alpha2A-adrenoceptor subtype.