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We analyzed the role of P/Q-type calcium channels in sciatic nerve regeneration after lesion induced by chronic constriction injury (CCI) in heterozygous null mutant mice lacking the CaV2.1α1 subunit of these channels (Cacna1a+/-). Compared with wild type, Cacna1a+/- mice showed an initial reduction of the CCI-induced allodynia, indicating a reduced pain perception, but they also evidenced a lack of recovery over time, with atrophy of the injured hindpaw still present 3 months after CCI when wild-type mice fully recovered. In parallel, Cacna1a+/- mice exhibited an early onset of age-dependent loss of P/Q-type channels, which can be responsible for the lack of functional recovery. Moreover, Cacna1a+/- mice showed an early age-dependent reduction of muscular strength, as well as of Schwann cells proliferation and sciatic nerve remyelination. This study demonstrates the important role played by P/Q-type channels in recovery from nerve injury and has important implications for the knowledge of age-related processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The use of botulinum neurotoxin type A (BoNT/A) against pain, with emphasis for its possible use in alleviating chronic pain, still represents an outstanding challenge for experimental research. In this study, we examined the effects of BoNT/A on morphine-induced tolerance during chronic morphine treatment in neuropathic CD1 mice subjected to sciatic nerve lesion according to the Chronic Constriction Injury (CCI) model of neuropathic pain. We measured the effects of BoNT/A on CCI-induced allodynia and hyperalgesia and on the expression of glial fibrillary acidic protein (GFAP, marker of astrocytes), complement receptor 3/cluster of differentiation 11b (CD11b, marker of microglia), and neuronal nuclei (NeuN) at the spinal cord level. We also analyzed the colocalized expression of GFAP, CD11b and NeuN with phosphorylated p-38 mitogen-activated protein kinase and with μ-opioid receptor (MOR). A single intraplantar injection of BoNT/A (15 pg/paw) into the injured hindpaw, the day before the beginning of chronic morphine treatment (9 days of twice daily injections of 40 mg/kg morphine), was able to counteract allodynia and enhancement of astrocytes expression/activation induced by CCI. In addition, BoNT/A increased the analgesic effect of morphine and countered morphine-induced tolerance during chronic morphine treatment. These effects were accompanied, in neurons, by re-expression of MORs that had been reduced by repeated morphine administration. The combinatory effects of BoNT/A and morphine could have relevant therapeutic implications for sufferers of chronic pain who could benefit of pain relief reducing tolerance due to repeated treatment with opiates., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Over the recent years compelling evidence has accumulated indicating that botulinum neurotoxin serotype A (BoNT/A) results in analgesic effects on neuropathic as well as inflammatory pain, both in humans and in animal models. In the present study, the pharmacological interaction of BoNT/A with morphine in fighting inflammatory pain was investigated in mice using the formalin test. Moreover, the effects of BoNT/A on the tolerance-induced by chronic administration of morphine were tested and the behavioral effects were correlated with immunofluorescence staining of glial fibrillary acidic protein, the specific marker of astrocytes, at the spinal cord level. An ineffective dose of BoNT/A (2 pg/paw) combined with an ineffective dose of morphine (1 mg/kg) exerted a significant analgesic action both during the early and the late phases of formalin test. A single intraplantar injection of BoNT/A (15 pg/paw; i.pl.), administered the day before the beginning of chronic morphine treatment (7 days of s.c. injections of 20 mg/kg), was able to counteract the occurrence of tolerance to morphine. Moreover, BoNT/A reduces the enhancement of the expression of astrocytes induced by inflammatory formalin pain. Side effects of opiates, including the development of tolerance during repeated use, may limit their therapeutic use, the possibility of using BoNT/A for lowering the effective dose of morphine and preventing the development of opioid tolerance would have relevant implications in terms of potential therapeutic perspectives., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nerve Growth Factor (NGF) signalling is mediated by the TrkA and p75NTR receptors. Besides its neurotrophic and survival activities, NGF displays a potent pro-nociceptive activity. Recently, a missense point mutation was found in the NGFB gene (C661T, leading to the aminoacid substitution R100W) of individuals affected by a form of hereditary loss of pain perception (hereditary sensory and autonomic neuropathy type V, HSAN V). In order to gain insights into the functional consequences of the HSAN V NGF mutation, two sets of hNGFR100 mutants were expressed in Escherichia coli and purified, as mature NGF or proNGF, for in vitro receptor binding studies. Here, we show by Surface Plasmon Resonance analysis that the R100 mutation selectively disrupts binding of hNGF to p75NTR receptor, to an extent which depends on the substituting residue at position 100, while the affinity of hNGFR100 mutants for TrkA receptor is not affected. As for unprocessed hproNGF, the binding of the R100 variants to p75NTR receptor shows only a limited impairment, showing that the impact of the R100 mutation on p75NTR receptor binding is greater in the context of mature, processed hNGF. These results provide a basis for elucidating the mechanisms underlying the clinical manifestations of HSAN V patients, and provide a basis for the development of "painless" hNGF molecules with therapeutic potential., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objective: Biallelic ablation of VGF determines a dwarf phenotype. VGF precursor protein encodes for different biologically active peptides none of which has been related to growth or muscular abnormalities. Here we present the first attempt to fill this gap. We tested the hypothesis that a recently identified VGF-derived peptide, TLQP-21, shown to centrally modulate metabolic functions, could also modulate growth hormone (GH)-axis and muscle strength., Design: Adult male mice were chronically icv injected with TLQP-21 (15 microg/day for 14 days). Physiological, molecular and behavioral parameters related to the GH/IGF-1-axis were investigated., Results: Except for a reduction in the soleus weight, TLQP-21 did not affect GH/IGF-1-axis mediators, muscle strength and muscle weight., Conclusions: Results collected exclude a role for TLQP-21 in modulating the GH/IGF1-axis and muscle functions. VGF-derived peptides involved in the dwarf phenotype of VGF-/- mice have to be identified yet.

Stressful events during certain neonatal periods may increase the vulnerability of an individual to develop psychopathology and/or drug dependence later in life. Therefore, in the present study, we assessed activity levels, emotionality, sensitivity to the effects of morphine, as well as expression of proenkephalin and prodynorphin in several brain regions in 35 and 90-day-old male mice, subjected to postnatal manipulation consisting in brief exposures to clean bedding (CB). In comparison with controls, CB mice showed reduced emotionality expressed as percentage of time in open arms of the elevated plus maze both at 35 days of life and in adulthood. Increased nociceptive threshold was also present in both time points measured. Conversely, higher locomotor activity was recorded in 35 days of life but not in adulthood. Analysis of film autoradiograms revealed no changes in prodynorphin mRNA level, but statistically significant decrease in the level of proenkephalin mRNA in striatum in young CB mice in comparison with young controls; no difference was observed between adult CB and control animals. CB adult mice also showed hypersensitivity to the rewarding effect of morphine in comparison with controls in the place preference test. In conclusion, our results revealed that in the critical period of development the effects of manipulation were evident, not only on behavioral responses but also on the neurochemical markers considered in the present research. Postnatal manipulation could induce changes in the dynamic neuronal processes occurring during development with long-term behavioral effects.

The effects of the dihydropyridine calcium channel blocker nimodipine on avoidance impairment induced by the tricyclic antidepressant amitriptyline were assessed during shuttle-box training and in previously trained mice of the DBA/2 strain. Nimodipine (0, 0.5, 1, 2.5, or 5 mg/kg) had no effect alone, but attenuated the avoidance impairment induced by 5 mg/kg amitriptyline on avoidance acquisition, as well as on a previously learned avoidance response. The avoidance improving action of the calcium channel blocker was less evident in mice receiving a larger dose (7.5 mg/kg) of the antidepressant drug. The effect of nimodipine did not appear to be specifically related to the avoidance impairment induced by amitriptyline, because the calcium antagonist also attenuated the avoidance impairing action of the neuroleptic chlorpromazine. The avoidance impairment induced by amitriptyline and chlorpromazine, and the related ameliorating action of nimodipine, seem imputable to drug effects on the performance of the avoidance response, rather than to interferences with learning processes. The results suggest that, in the case of concomitant administration, nimodipine could alleviate adverse side effects of tricyclic antidepressant, i.e., psychomotor disturbances.

The present research was undertaken to investigate, by behavioral and immunohistochemical methods, the effects of intrathecal (i.th.) injection of the muscarinic agonist oxotremorine on the response to the long-lasting nociceptive stimulus induced by injection of formalin into the rat hind paw. Formalin injection induced a biphasic, pain-induced behavioral response (paw jerks), as well as an increase in the number of nitric oxide (NO) synthase-labeled neurons in laminae I-III, IV, and X, but not in laminae V-VI. Oxotremorine (0.1-10 ng, i.th.) inhibited paw-jerk frequency in both phases of formalin-induced behavior. The immunohistochemical results showed that i.th.-injected oxotremorine differently affected the level of NO synthase in lumbar part of the spinal cord: no change or increase after the dose of 1 ng, and a significant reduction of nitric oxide synthase neurons after the higher dose (10 ng). These results evidenced a role of cholinergic system in the modulation of tonic pain and in nitric oxide synthase expression at the spinal cord level, which further suggests that these two systems could be involved in phenomena induced by long-lasting nociceptive stimulation.

The present study investigated the role of spinal nitric oxide (NO) in the antinociception induced by intraperitoneal (i.p.) and intrathecal (i.th.) injection of oxotremorine. The experiments were carried out on male Wistar rats, which had cannulas chronically implanted in the lumbar enlargement of the spinal cord. Antinociceptive effects were evaluated using a tail-flick and a paw pressure test. To raise the spinal NO level, the rats received the NO donor, 3-morpholino-sydnonimine (SIN-1, 10 and 100 microg/5 microl); to lower the NO level, the inhibitor of NO synthase, N-nitro-L-arginine methyl ester (L-NAME, 50 and 400 microg/5 microl), was administered. Both those substances were injected i.th. Systemic injections of oxotremorine (0.02 and 0.1 mg/kg) produced a significant increase in the thermal nociceptive threshold, while the mechanical threshold was affected only by the higher dose (0.1 mg/kg) of the muscarinic agonist. I.th. injections of oxotremorine (0.1 ng, 1 ng, 1 microg/5 microl) produced significant antinociception in both those tests. I.th. administration of SIN-1 in doses which themselves did not affect the nociceptive threshold antagonized both the peripheral and central oxotremorine antinociception. I.th. administration of L-NAME (50 and 400 microg/5 microl) did not change the nociceptive threshold, but dose-dependently potentiated the effects of oxotremorine injected i.p. in both tests; however, the effect of i.th. administration of oxotremorine was potentiated only in the tail-flick test. Our results demonstrate that irrespective of the way of its injection, the antinociceptive effect of oxotremorine is modulated by activity of the spinal NO. Moreover, our results further support the hypothesis that NO present in the spinal cord exerts pronociceptive effects.

Dominant and subordinate male mice behave differently when exposed to a female, with subordinates showing impairment of their sexual performance in the presence of the male antagonist. In the present study, we investigate whether these rank-related behavioral differences can be modified by an anxiolytic treatment. In a first experiment, diazepam (0.25 mg/kg) improves the performance of subordinate mice toward the female, as shown by the increase of proxemic behavior, anogenital sniffing, and social grooming of the female. Social grooming of the female is the only behavior modified by a higher dose of the anxiolytic drug (0.5 mg/kg). A second experiment, in which dominant and isolated mice are subjected to the same experimental procedure, demonstrates that social behavior of these two classes of males is not affected by the pharmacological treatment. The results are discussed in terms of the advantages of using subordinate males in such a sexual context as a model for the study of anxiolytic drugs.

Subhypnotic doses (10 and 20 mg/kg) of pentobarbital significantly elevated locomotor activity measured for 30 min in CD-1 mice. The hyperactivity was also observed in mice recovering from pentobarbital-induced (50 mg/kg) sleep (measurements starting 15 min after recovery of righting reflex). Naloxone in doses up to 4 mg/kg did not affect significantly the pentobarbital-induced hyperactivity in any experiment; a dose of 8 mg/kg only partially attenuated the hyperactivity induced by a dose of 20 mg/kg of pentobarbital, but did not affect significantly either the stimulatory effect of a low subhypnotic dose (10 mg/kg) or the posthypnotic hyperactivity. This suggests a negligible involvement of opioid mechanisms in the hyperactivity induced by pentobarbital.

A passive avoidance methodology was used to test the effect of ethanol, and its interference with GABAergic mechanisms, on memory in male CD1 mice. Retention performance was reduced in a dose-related manner, by ethanol and by muscimol, a GABA agonist, while it was increased by the GABA antagonists picrotoxin and bicuculline. These effects were evident when treatments were carried out immediately, but not 120 min, after training, suggesting that they were due to a specific action of the drugs on the time-dependent memory consolidation process. The ethanol-induced reduction of retention performance was enhanced by muscimol and decreased by picrotoxin and bicuculline administrations. Taken together the results confirm the involvement of a GABAergic mechanism in memory consolidation and demonstrate that it underlies the negative effect of ethanol on passive avoidance behavior in the mouse.

Classical conditioning of stress-induced analgesia (SIA) resulted in higher tail flick latencies in BALB/c mice. Transfer of classical conditioning of SIA was evident when the same repetition rate (pulsed light or pulsed tone) characterized stimuli in different sensory modalities. This finding is discussed in terms of opioid production and of generalization of emotional reactions.

The synthesis of a series of physostigmine analogs, in which the methylcarbamyl group has been substituted with monoalkylcarbamyl, dimethyl- and diethylcarbamyl groups, is reported. These compounds were prepared with the aim of investigating their possible therapeutic effects in the treatment of Alzheimer's type dementia. The new analogs of physostigmine are inhibitors of acetylcholinesterase from Electroforus electricus, with a value of the reactivation constant, k3 smaller than the one of physostigmine. The percentage of anticholinesterase activity in vitro and in vivo, the acute toxicity and some behavioural effects were also evaluated for selected derivatives. The reactivation constant, in vitro, supports the view that the derivatives described would be more suitable for therapeutic use than physostigmine.

The involvement of GABAergic mechanisms in the effects exerted by the opioid antagonists naloxone and naltrexone on memory consolidation was investigated in CD1 mice tested in a one-trial inhibitory avoidance task. In a first group of experiments posttraining administration of naloxone (2.0 and 4.0 but not 1.0 mg/kg) and naltrexone (0.5 and 1.0 but not 0.25 mg/kg), as well as those of the GABA-antagonists picrotoxin (0.5 and 1.0 but not 0.25 mg/kg) and bicuculline (0.25 and 0.5 but not 0.1 mg/kg) enhanced, whereas those of the GABA-agonist muscimol (1.0 and 2.0 but not 0.5 mg/kg) impaired retention on a 24-hr test. In a second group of experiments, picrotoxin, or bicuculline, administration enhanced, while muscimol treatment attenuated the effects of naloxone and naltrexone on retention. The results suggest that naloxone and naltrexone may influence memory consolidation in CD1 mice by interacting with the GABAergic system.

A series of analogues of physostigmine were prepared with the aim of investigating their inhibitory effects on acetylcholinesterase in the treatment of Alzheimer's disease. One of the isomers prepared was evaluated for its anticholinesterase activity in vivo, acute toxicity, and some behavioral effects. This compound was a competitive inhibitor of the enzyme and was found to antagonize the stimulating effect produced by scopolamine on locomotor activity and to facilitate memory consolidation.