Alessandro Lecci

A model to measure anticipatory anxiety in mice

Among animals from the same cage, mice removed last had a higher temperature compared to those removed first. This phenomenon a) persisted 2 and 24 h later; b) was present regardless of the number of the animals (5, 10, 15 and 20) in each cage, c) was independent of whether the number of animals was reduced or maintained constant in the cage and d) could even be observed by reversing the order of removal of the animals from the cage. In addition, the fewer the animals allocated to a cage the greater the percentage of those which became hyperthermic. This rise in rectal temperature of mice removed last was prevented by diazepam (2.5 and 5 mg/kg PO, 30 min), nitrazepam (2 and 4 mg/kg PO, 30 min) but not by imipramine (15 and 30 mg/kg PO, 30 min) or haloperidol (0.5 and 1 mg/kg PO, 60 min) and was observed in a greater opercentage of mice following subcutaneous yohimbine treatment (2 mg/kg, 60 min). This phenomenon does not seem to depend on physical exercise due to an attempt to escape, since no correlation appears to exist between motor activity (open-field) and rise in rectal temperature. These data would seem to indicate that hyperthermia in the last animals may represent a new tool for studying the neurobiology of anticipatory(?) anxiety.

Cyclophosphamide cystitis in rats: involvement of capsaicin-sensitive primary afferents

The involvement of capsaicin-sensitive primary afferent neurons in cyclophosphamide (CYP)-induced cystitis has been investigated in rats. CYP (150 mg/kg) was administered 48 h before testing in both vehicle- and capsaicin- (50 mg/kg s.c., 4 days before) treated rats. Some experiments were also performed 96 h after bilateral removal of pelvic ganglia to produce bladder denervation. CYP administration produced a marked detrusor hyperreflexia which was abolished by capsaicin pretreatment, demonstrating that it is mediated through stimulation of capsaicin-sensitive afferent neurons. CYP administration was followed by a marked increase in bladder weight and plasma protein extravasation (measured by the Evans blue leakage technique). The latter effect was largely prevented by ganglionectomy but was aggravated by capsaicin pretreatment. The effect of capsaicin was suppressed by ganglionectomy. Isolated strips of detrusor muscle from CYP-treated animals developed less tension in response to various stimuli as compared to strips from vehicle-treated animals; however, when contractile responses were expressed as percentage of an internal standard (carbachol-induced contraction) no difference was evident between the two groups. The bladder content of calcitonin gene-related peptide, used as a marker of the bladder afferent fibres that are capsaicin-sensitive in adult rats, was slightly reduced as compared to controls, but the difference can be accounted for by the increased bladder weight. We conclude that CYP-induced cystitis is not accompanied by a toxic effect on bladder nerves and that the decrease in bladder capacity is entirely mediated through stimulation of capsaicin-sensitive afferent fibres, presumably linked to the formation of the irritant metabolite of CYP, acrolein.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological validation of a novel animal model of anticipatory anxiety in mice

The current study investigates the action of anxiolytics, antidepressants, neuroleptics, antipyretics, muscle relaxants, antihypertensives and naloxone in a novel animal model of anxiety, based on the evidence that mice removed last from their cage develop hyperthermia (stress-induced hyperthermia, SIH) when compared to those removed first. Alprazolam (0.15–0.6 mg/kg), chlordiazepoxide (25 mg/kg), estazolam (1 mg/kg), phenobarbital (20 mg/kg), ethanol (2 and 4 g/kg), buspirone (5 and 10 mg/kg) and prazosin (1 and 2 mg/kg), as well as repeatedly administered diazepam (5 mg/kg), inhibited SIH. In contrast, tofisopam (12.5–200 mg/kg), desipramine (15 and 30 mg/kg), amitriptyline (10 mg/kg), fluoxetine (10 and 20 mg/kg), tranylcypromine (5 and 10 mg/kg), chlorpromazine (1 and 2 mg/kg), clozapine (2 and 4 mg/kg), pimozide (0.5 and 1 mg/kg),l-sulpiride (15 and 30 mg/kg),l-propranolol (5 and 10 mg/kg), acetyl salicylic acid (200 and 400 mg/kg), indomethacin (2.5 and 5 mg/kg), verapamil (2.5 and 5 mg/kg), captopril (25 and 50 mg/kg), dantrolene (10 and 20 mg/kg), mephenesin (300 and 600 mg/kg),d-amphetamine (1 and 4 mg/kg) and naloxone (2.5 and 15 mg/kg) were inactive, as were 10 mg/kg imipramine, amitriptyline and fluoxetine injected every day for 21 days. Reserpine at high doses (1.25 and 2.5 mg/kg) but not at a lower dose (0.62 mg/kg) prevented SIH, but in this case animals showed a behavioural syndrome which could have interfered with the occurrence of the hyperthermia.

Discovery of antidepressant activity by forced swimming test may depend on pre-exposure of rats to a stressful situation

Antidepressant-induced anti-immobility effects have been assessed in animals exposed or not to a pretest session using the forced swimming test. Desipramine, maprotiline, mianserine (15 and 30 mg/kg), nomifensine (2.5 and 5 mg/kg), d-amphetamine (1 and 2 mg/kg) and muscimol (1 and 2 mg/kg), unlike imipramine (15 and 30 mg/kg), LY-171555 (0.1 and 0.2 mg/kg) and scopolamine (0.5 and 0.1 mg/kg), did not reduce immobility time in rats which had not received the pretest session. On the other hand, all of the drugs tested reduced immobility time in rats exposed to a pretest session. In addition, the degree of anti-immobility effects of desipramine (20 mg/kg) and nomifensine (5 mg/kg) increased proportionally with the level of water (0, 4, 15 and 30 cm) to which animals were exposed at the time of pretest. Furthermore, desipramine reduced immobility time in rats pre-exposed to types of stress different from forced swimming, cold, restraint or foot-shock. All drugs were injected intraperitoneally three times, 24, 5 and 1 h before testing. The present findings suggest that a stressful pretest session may reveal pharmacological properties of antidepressants in the forced swimming test. This is also substantiated by the fact that diazepam (2.5 and 5 mg/kg) administered 30 min before the swimming pretest antagonized the anti-immobility effect of 15 mg/kg desipramine.

Pharmacological profile of the novel mammalian tachykinin, hemokinin 1

The effects of the novel mammalian tachykinin, hemokinin 1 (HEK‐1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. Similar to SP (Ki=0.13 nM), HEK‐1 inhibited in a concentration‐dependent manner and with high affinity [3H]‐substance P (SP) binding to human NK1 receptor (Ki=0.175 nM) while its affinity for [125I]‐neurokinin A (NKA) binding at human NK2 receptor was markedly lower (Ki=560 nM). In isolated bioassays HEK‐1 was a full agonist at tachykinin NK1, NK2 and NK3 receptors. In the rat urinary bladder (RUB) HEK‐1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK‐1 and in the guinea‐pig ileum (GPI), HEK‐1 was about 500 fold less potent than NKA and NKB, respectively. The responses to HEK‐1 were antagonized by GR 82334 in RUB (pKB=5.6±0.07), by nepadutant in RPA (pKB=8.6±0.04) and by SR 142801 in GPI (pKB=9.0±0.2) with apparent affinities comparable to that measured against tachykinin NK1, NK2 and NK3 receptor‐selective agonists, respectively. Intravenous HEK‐1 produced dose‐related decrease of blood pressure in anaesthetized guinea‐pigs (ED50=0.1 nmol kg−1) and salivary secretion in anaesthetized rats (ED50=6 nmol kg−1) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK1 receptor antagonist, SR 140333. We conclude that HEK‐1 is a full agonist at tachykinin NK1, NK2 and NK3 receptors, possesses a remarkable selectivity for NK1 as compared to NK2 or NK3 receptors and acts in vivo experiments with potency similar to that of SP.

Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder

1 Cyclophosphamide (CYP) (150 mg kg−1, i.p. 0.5–48 h before) caused a time‐dependent plasma protein extravasation in the rat urinary bladder with the maximal extravasation occuring at between 2 and 4 h after administration of the drug. 2 Prior capsaicin desensitization of capsaicin‐sensitive primary afferent neurones (CSPANs) (50 mg kg−l, s.c., 4 days before) resulted in approximately 50% inhibition of the magnitude of the extravasation response at the 2 h time‐point. 3 Intraperitoneal (i.p.) pretreatment with the tachykinin NK1 receptor antagonist, RP 67,580 (0.44 mg kg−1) or the bradykinin B2 receptor antagonist, Hoe 140 (0.13 mg kg−1) had significant inhibitory effects, giving responses of 56 ± 6% and 39 ± 4% of the control extravasation response to CYP treatment after 2 h. Pretreatment with the tachykinin NK2 receptor antagonist, SR 48,968 (0.3 mg kg−1, i.p.), the histamine H1 receptor blocker, chlorpheniramine (10 mg kg−1, i.p.), the 5‐HT receptor blocker, methysergide (6 mg kg−l, i.p.) or the cyclo‐oxygenase inhibitor indomethacin (5 mg kg−1, i.p.) had no significant effect upon the development of the extravasation response at this same time‐point. 4 In rat isolated urinary bladder strips, the active metabolite of CYP, acrolein (1–300 μm) produced a concentration‐dependent contraction that was significantly reduced by in vitro capsaicin desensitization (10 μm for 15 min) indicating direct stimulation of CSPANs. CYP was without appreciable effect. 5 The effect of acrolein in vitro was significantly reduced by pretreatment of the bladder with a combination of tachykinin NK1 and NK2 receptor antagonists, RP 67,580 (3 μm) and SR 48,968 (1 μm). The dose‐response curve to acrolein was also significantly inhibited by treatment with indomethacin (10 μm) and slightly affected by Hoe 140 (1 μm). 6 These findings demonstrate the contribution of CSPANs to the development of CYP‐induced cystitis. Plasma protein extravasation involves activation of tachykinin NK1 and bradykinin B2 receptors. Activation of CSPANs in the urinary bladder is likely to be due to the conversion of CYP into its active metabolite, acrolein, and not to a direct effect of CYP upon these nerve‐endings.

Effect of nociceptin on heart rate and blood pressure ananaesthetized rats

We now report on the effects of nociceptin, the endogenous ligand for the orphan opioid-like ORL1 receptor, on cardiovascular parameters (blood pressure and heart rate) in urethane-anaesthetized rats. Nociceptin dose dependently (10-100 nmol/kg i.v.) produced a transient (< 10 min) hypotension and bradycardia. The reduction in blood pressure was inhibited by guanethidine pretreatment and unaffected by bilateral cervical vagotomy. The bradycardia elicited by nociceptin was reduced after bilateral vagotomy and by guanethidine and was abolished by the combination of the two treatments. These findings indicate that nociceptin exerts a pronounced depressant effect on cardiovascular function which is produced indirectly through a concomitant inhibition and activation, respectively, of the sympathetic and parasympathetic outflows to the cardiovascular system.

Pharmacology of transmission to gastrointestinal muscle.

The identity of excitatory and inhibitory neurotransmitters is well established. Excitatory motor neurons synthesize and release acetylcholine and tachykinins, which act through postjunctional muscarinic M2 and M3 or tachykinin NK1 and NK2 receptors, respectively, to induce smooth muscle contraction. A residual excitatory component is mediated by ATP acting on P2X1 receptors. Conversely, inhibitory motor neurons express nitric oxide synthase and vasoactive intestinal peptide (VIP), which together with ATP, induce a coordinated muscle relaxation. The receptors involved in the inhibitory effects of ATP and VIP are unknown. Likewise, the relationships between inhibitory signals triggered by NO and those mediated by VIP need to be clarified. Recent evidence obtained using receptor knockout mice have confirmed the involvement of the above-mentioned excitatory transmitters but have revealed an unexpected complexity in the nitrergic transmission, where the effects of NO are manifested only in the presence of carbon monoxide. Interstitial cells of Cajal (ICC) are being recognized as targets of intestinal motor neurons; therefore, the signaling mechanisms are probably integrated by these cells before being transmitted to smooth muscle. Challenges in future years will be to identify the physiological role of the various excitatory and inhibitory components, and to understand the relative importance of neurotransmitter receptors expressed on ICC and smooth muscle cells.

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist

The pharmacological profile was studied of MEN 11420, or cyclo{[Asn(β‐D‐GlcNAc)‐Asp‐Trp‐Phe‐Dap‐Leu]cyclo(2β‐5β)}, a glycosylated derivative of the potent, selective, conformationally‐constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met‐Asp‐Trp‐Phe‐Dap‐Leu)cyclo(2β‐5β)). MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]‐neurokinin A and [3H]‐SR 48968 with Ki values of 2.5±0.7 nM (n=6) and 2.6±0.4 nM (n=3), respectively. MEN 11420 showed negligible binding affinity (pIC50<6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor‐mediated contractions (pKB=8.6±0.07, n=10, and 9.0±0.04, n=12; Schild plot slope=−1.06 (95% c.l.=−1.3; −0.8) and −1.17 (95% c.l.=−1.3; −1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pKB of 10.2±0.14, n= 9, and 9.8±0.15, n=9, was calculated in the hamster trachea and mouse urinary bladder, respectively. MEN 11420 showed low affinity (pKB<6) at guinea‐pig and rat tachykinin NK1 (guinea‐pig ileum and rat urinary bladder) and NK3 (guinea‐pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. The in vivo antagonism of the contractions produced by [βAla8]neurokinin A(4–10) (1 nmol kg−1) was observed after intravenous (dose range: 1–10 nmol kg−1), intranasal (3–10 nmol kg−1), intrarectal (30–100 nmol kg−1) and intraduodenal (100–300 nmol kg−1) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. A dose of MEN 11420 (100 nmol kg−1, i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [βAla8]neurokinin A(4–10) (10–300 nmol kg−1), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1–10 nmol kg−1). These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Role of NK1 tachykinin receptors in thermonociception: Effect of (±)-CP 96,345, a non-peptide substance P antagonist, on the hot plate test in mice

We have tested the ability of (+/-)-CP 96,345, a novel nonpeptide substance P (SP) antagonist, to block the aversive behaviour induced by intrathecal (i.t.) administration of SP and to induce thermal antinociception in mice. (+/-)-CP 96,345 administered i.t. or i.p. selectively blocked the effect of i.t. SP while leaving the response to i.t. bombesin unaffected. At the same dose proven effective against i.t. SP, (+/-)-CP 96,345 produced thermal analgesia in the hot plate test (52 degrees C). Using isolated organs for bioassay evaluation of activity at tachykinin receptor, (+/-)-CP 96,345 was found to be a potent (pA2 8.11, c.l. 7.9-8.3) and competitive NK1 receptor antagonist while it was devoid of activity at NK2 or NK3 receptors. These findings provide clear indication for the participation of SP, via NK1 receptors, in thermal nociception.