Increase of neurofilament-H protein in sensory neurons in antiretroviral neuropathy: Evidence for a neuroprotective response mediated by the RNA-binding protein HuD.

Nucleoside reverse transcriptase inhibitors (NRTIs) are key components of HIV/AIDS treatment to reduce viral load. However, antiretroviral toxic neuropathy has become a common peripheral neuropathy among HIV/AIDS patients leading to discontinuation of antiretroviral therapy, for which the underlying pathogenesis is uncertain. This study examines the role of neurofilament (NF) proteins in the spinal dorsal horn, DRG and sciatic nerve after NRTI neurotoxicity in mice treated with zalcitabine (2',3'-dideoxycitidine; ddC). ddC administration up-regulated NF-M and pNF-H proteins with no effect on NF-L. The increase of pNF-H levels was counteracted by the silencing of HuD, an RNA binding protein involved in neuronal development and differentiation. Sciatic nerve sections of ddC exposed mice showed an increased axonal caliber, concomitantly to a pNF-H up-regulation. Both events were prevented by HuD silencing. pNF-H and HuD colocalize in DRG and spinal dorsal horn axons. However, the capability of HuD to bind NF mRNA was not demonstrated, indicating the presence of an indirect mechanism of control of NF expression by HuD. RNA immunoprecipitation experiments showed the capability of HuD to bind the BDNF mRNA and the administration of an anti-BDNF antibody prevented pNF-H increase. These data indicate the presence of a HuD - BDNF - NF-H pathway activated as a regenerative response to the axonal damage induced by ddC treatment to counteract the antiretroviral neurotoxicity. Since analgesics clinically used to treat neuropathic pain are ineffective on antiretroviral neuropathy, a neuroregenerative strategy might represent a new therapeutic opportunity to counteract neurotoxicity and avoid discontinuation or abandon of NRTI therapy.

Chitinase 3-like proteins as diagnostic and prognostic biomarkers of multiple sclerosis.

BACKGROUND: Despite sensitivity of MRI to diagnose multiple sclerosis (MS), prognostic biomarkers are still needed for optimized treatment. OBJECTIVE: The objective of this paper is to identify cerebrospinal fluid (CSF) diagnostic biomarkers of MS using quantitative proteomics and to analyze their expression at different disease stages. METHODS: We conducted differential analysis of the CSF proteome from control and relapsing-remitting MS (RRMS) patients followed by verification by ELISA of candidate biomarkers in CSF and serum in control, clinically isolated syndrome (CIS), RRMS and progressive MS (PMS) patients. RESULTS: Twenty-two of the 527 quantified proteins exhibited different abundances in control and RRMS CSF. These include chitinase 3-like protein 1 (CHI3L1) and 2 (CHI3L2), which showed a strong expression in brain of MS patients, especially in astrocytes and microglial cells from white matter plaques. CSF and serum CHI3L1 levels increased with the disease stage and CIS patients with high CSF (>189 ng/ml) and serum (>33 ng/ml) CHI3L1 converted more rapidly to RRMS (log rank test, p < 0.05 and p < 0.001, respectively). In contrast, CSF CHI3L2 levels were lower in PMS than in RRMS patients. Accordingly, CSF CHI3L1/CHI3L2 ratio accurately discriminated PMS from RRMS. CONCLUSIONS: CSF CHI3L1 and CHI3L2 and serum CHI3L1 might help to define MS disease stage and have a prognostic value in CIS.

PKC-mediated HuD-GAP43 pathway activation in a mouse model of antiretroviral painful neuropathy.

Patients treated with nucleoside reverse transcriptase inhibitors (NRTIs) develop painful neuropathies that lead to discontinuation of antiretroviral therapy thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathy are not known. In order to elucidate the mechanisms underlying this drug-induced neuropathy, we have characterized cellular events in the central nervous system following antiretroviral treatment. Systemic administration of the antiretroviral agent, 2',3'-dideoxycytidine (ddC) considerably increased the expression and phosphorylation of protein kinase C (PKC) γ and ɛ, enzymes highly involved in pain processes, within periaqueductal grey matter (PAG), and, to a lesser extent, within thalamus and prefrontal cortex. These events appeared in coincidence with thermal and mechanical allodynia, but PKC blockade did not prevent the antiretroviral-induced pain hypersensitivity, ruling out a major involvement of PKC in the ddC-induced nociceptive behaviour. An increased expression of GAP43, a marker of neuroregeneration, and decreased levels of ATF3, a marker of neuroregeneration, were detected in all brain areas. ddC treatment also increased the expression of HuD, a RNA-binding protein target of PKC known to stabilize GAP43 mRNA. Pharmacological blockade of PKC prevented HuD and GAP43 overexpression. Silencing of both PKCγ and HuD reduced GAP43 levels in control mice and prevented the ddC-induced GAP43 enhanced expression. Present findings illustrate the presence of a supraspinal PKC-mediated HuD-GAP43 pathway activated by ddC. Based on our results, we speculate that antiretroviral drugs may recruit the HuD-GAP43 pathway, potentially contributing to a response to the antiretroviral neuronal toxicity.

Pharmacological in vivo test to evaluate the bioavailability of some St John's Wort innovative oral preparations.

In this study, the optimisation of biopharmaceutical properties of a dried commercial extract of St John's Wort were evaluated employing the in vivo forced swimming test (FST). Three new dosage forms containing beta-cyclodextrin and surfactants (SDS, ASC8) were compared in the FST with the commercial extract. The commercial extract showed antidepressant activity in mice after 60 min at a dosage of 100 mg/kg. The same antidepressant activity appeared in 30 min with a micellar solution of SDS containing the same quantity of extract (100 mg/kg), while with micelles of ASC8 the effect appeared at 15 min and with a dosage of 30 mg/kg. In the case of beta-cyclodextrin the best results were obtained at 30 min, administering 60 mg/kg of the extract. Finally, the influence of the formulations on the water solubility of the constituents of the extract is reported. The tensides dramatically enhanced solubility, in particular that of the more lipophilic compounds, in the case of beta-cyclodextrin this effect was very pronounced for flavonoids and biapigenin, lower for hypericins and practically insignificant for hyperforins.

An antidepressant behaviour in mice carrying a gene-specific InsP3R1, InsP3R2 and InsP3R3 protein knockdown.

Evidence has accumulated for the involvement of Ca(2+) in the pathophysiology of mood disorders. Elevations in both resting and stimulated intracellular Ca(2+) levels in patients with affective disorders have been reported. The role of inositol-1,4,5-trisphosphate receptors (InsP3Rs), which allow mobilization of intracellular Ca(2+) stores, was, then, investigated in the mouse forced swimming test. InsP3R antagonists (heparin, xestospongin C) as well as an inositol monophosphatase inhibitor (LiCl) showed an antidepressant activity of intensity comparable to clinically used antidepressants. InsP3Rl, InsP3R2 and InsP3R3 knockdown mice were obtained to investigate the role of InsP3R isoforms. We generated mice carrying a cerebral knockdown of InsP3Rl, InsP3R2 and InsP3R3 proteins by administering antisense oligonucleotides complementary to the sequence of InsP3Rl, InsP3R2 and InsP3R3. These antisense-treated mice showed a specific InsP3R protein level reduction in the mouse cerebral cortex and hippocampus, demonstrated by immunoblotting, immunoprecipitation and immunocytochemistry experiments. Knockdown mice for each InsP3R isoforms showed an antidepressant behaviour and the induced phenotype was reversible disappearing 7 days after the end of the treatment. The absence of impairment of locomotor activity and spontaneous mobility in InsP3R knockdown mice was revealed. These results indicate the involvement of the InsP3R-mediated pathway in the modulation of depressive conditions and may be useful for the development of new therapeutical strategies for the treatment of mood disorders.

Knockdown of the type 2 and 3 inositol 1,4,5-trisphosphate receptors suppresses muscarinic antinociception in mice.

The involvement of central endoplasmic inositol 1,4,5-trisphosphate receptors (IP3R) in muscarinic antinociception was investigated in the mouse hot plate test. Selective knockdown of type 1, 2 and 3 IP3R was obtained by means of an antisense oligonucleotide (aODN) strategy. A selective IP3R protein level reduction of approximately 30-50% produced by aODN administration for each receptor subtype investigated was demonstrated by Western blotting experiments. I.c.v. pretreatment with an aODN complementary to the sequence of the type 2 IP3R (0.1-3 nmol per mouse i.c.v.) prevented the antinociception induced by physostigmine (0.15 mg kg(-1) s.c.) and oxotremorine (60 microg kg(-1) s.c.). Similarly, an aODN against type 3 IP3R (0.1-3 nmol per mouse i.c.v.) antagonized cholinergic antinociception. A shift to the right of the physostigmine dose-response curve was obtained after anti-type 2 IP3R2 and anti-type 3 IP3R treatments. Conversely, pretreatment with an aODN complementary to the sequence of type 1 IP3R (0.1-5 nmol per mouse i.c.v.) did not modify the antinociception induced by physostigmine and oxotremorine. Mice undergoing treatment with aODNs did not show any impairment of the locomotor activity, spontaneous motility and exploratory activity as revealed by the rota-rod and hole board tests. These results indicate a selective involvement of type 2 and 3 IP3R in central muscarinic antinociception in mice.

Behavioural manipulation in a grasshopper harbouring hairworm: a proteomics approach.

The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insect's responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshopper's central nervous system (CNS) by producing certain 'effective' molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the host's CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.

Spinal RyR2 pathway regulated by the RNA-binding protein HuD induces pain hypersensitivity in antiretroviral neuropathy.

The antiretroviral toxic neuropathy, a distal sensory polyneuropathy associated with antiretroviral treatment, is a frequently occurring neurological complication during treatment of patients with AIDS and often leads to discontinuation of antiretroviral therapy. The mechanisms by which antiretroviral drugs contribute to the development of neuropathic pain are not known. Using drugs that reduce intracellular calcium ions (Ca(2+)), we investigated the hypothesis that altered cytosolic Ca(2+) concentration contributes to the 2',3'-dideoxycytidine (ddC)-evoked painful neuropathy. Administration of ddC induced mechanical and cold allodynia, which were abolished by intrathecal administration of TMB-8, a blocker of Ca(2+) release from intracellular stores, and by ryanodine, a RyR antagonist. Treatment with the IP3R antagonist heparin prevented mechanical allodynia with no effect on thermal response. To further clarify the pathway involved, we investigated the role of HuD, a RNA binding protein involved in neuronal function. HuD silencing reverted both mechanical and cold allodynia inducing, a phenotype comparable to that of ryanodine-exposed mice. HuD binding to the RyR2 mRNA, the most abundant RyR isoform in the spinal cord, was demonstrated and RyR2 silencing prevented the ddC-induced neuropathic pain. A positive regulation of gene expression on CaMKIIα by HuD was also observed, but sequestration of CaMKIIα had no effect on ddC-induced allodynia. The present findings identify a spinal RyR2 pathway activated in response to ddC administration, involving the binding activity on RyR2 mRNA by HuD. We propose the modulation of the RyR2 pathway as a therapeutic perspective in the management of antiretroviral painful neuropathy.

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 role of potassium channels in antihistamine analgesia.

The effect of the administration of pertussis toxin as well as modulators of different subtypes of K+ channels on the antinociception induced by the H1-antihistamines pyrilamine, diphenhydramine and promethazine was evaluated in the mouse hot plate test. Pretreatment with pertussis toxin (0.25 microg/mouse i.c.v.) prevented pyrilamine, diphenhydramine and promethazine antinociception. The K(ATP) channel openers minoxidil and pinacidil potentiated the antinociception produced by the H1-antihistamines whereas the K(ATP) channel blocker gliquidone prevented the anti H1-induced analgesia. The Ca2+-gated K+ channel blocker apamin antagonized pyrilamine, diphenhydramine and promethazine analgesia. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K+ channel, at the dose of 3.0 nmol/single i.c.v. injection, never modified the antinociception induced by the H1-antihistamines in comparison with degenerate oligonucleotide (dODN)-treated mice. At the highest effective doses, none of the drugs used modified animals' gross behaviour nor impaired motor coordination, as revealed by the rota rod test. The present data demonstrate that both K(ATP) and Ca2+-gated K+ channels, contrary to voltage-gated K+ channel Kv1.1, represent an important step in the transduction mechanism underlying central antinociception induced by H1-antihistamines.

Antisense 'knockdowns' of M1 receptors induces transient anterograde amnesia in mice.

The effect on memory processes of inactivation of the M1 gene by an antisense oligodeoxyribonucleotide (aODN) was investigated in the mouse passive avoidance test. Mice received a single intracerebroventricular (i.c.v.) injection of M1 aODN (0.3, 1.0 or 2.0 nmol per injection), degenerated ODN (dODN) or vehicle on days 1, 4 and 7. An amnesic effect, comparable to that produced by antimuscarinic drugs, was observed 12, 24, 48 and 72 h after the last i.c.v. aODN injection, whereas dODN and vehicle, used as controls, did not produce any effect. Reduction in the entrance latency to the dark compartment induced by aODN disappeared 7 days after the end of aODN treatment, which indicates the absence of any irreversible damage or toxicity caused by aODN. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that a decrease in M1 mRNA levels occurred only in the aODN-treated group, being absent in all control groups. Furthermore, a reduction in M1 receptors was observed in the hippocampus of aODN-treated mice. Neither aODN, dODN nor vehicle produced any behavioral impairment of mice. These results indicate that the integrity and functionality of M1 receptors are fundamental in the modulation of memory processes.

Presynaptic cholinergic modulators as potent cognition enhancers and analgesic drugs. 2. 2-Phenoxy-, 2-(phenylthio)-, and 2-(phenylamino)alkanoic acid esters.

Further modifications of the leads ((R)-(+)-hyoscyamine and (p-chlorophenyl)propionic acid alpha-tropanyl ester), which show analgesic and nootropic activities as a consequence of increased central presynaptic ACh release, are reported. 2-Phenoxy- and 2-(phenylthio)alkanoic acid esters showed the best results. Several members of these classes possess analgesic properties which are comparable to that of morphine and at the same time are able to reverse dicyclomine-induced amnesia. Confirmation was found that the mechanism of action is due to an increase in ACh release at central muscarinic synapses and that both auto- and heteroreceptors controlling ACh release are very likely involved. According to the results obtained with (R)-(+)-hyoscyamine, analgesic activity is stereochemistry dependent, since the R-(+)-enantiomers are always more efficacious than the corresponding S-(-)-ones. On the basis of their potency and acute toxicity, compounds (+/-)-28 (SM21) and (+/-)-42 (SM32) were selected for further study.

Design, synthesis, and preliminary pharmacological evaluation of 1, 4-diazabicyclo[4.3.0]nonan-9-ones as a new class of highly potent nootropic agents.

Several 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones have been synthesized and tested in vivo on mouse passive avoidance test, to evaluate their nootropic activity. The results show that they represent a new class of nootropic drugs with a pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference. Among the compounds studied, 7 (DM 232) shows outstanding potency, being active at the dose of 0. 001 mg kg(-1) sc.

Molecular simplification of 1,4-diazabicyclo[4.3.0]nonan-9-ones gives piperazine derivatives that maintain high nootropic activity.

Several 4-substituted 1-acylpiperazines, obtained by molecular simplification of 4-substituted 1,4-diazabicyclo[4.3.0]nonan-9-ones, have been synthesized and tested in vivo on the mouse passive avoidance test, to evaluate their nootropic activity. The results show that, apparently, an N-acylpiperazine group can mimic the 2-pyrrolidinone ring of 1,4-diazabicyclo[4.3.0]nonan-9-one, as the compounds of the new series maintain high nootropic activity. Moreover molecular simplification produces more clear-cut structure-activity relationships with respect to the parent series. The mechanism of action also appears to be similar in the two series. In fact, although the molecular mechanism remains to be elucidated, the most potent compound of each class (DM232 and 13, DM235) is able to increase acetylcholine release in rat brain. Piperazine derivatives represent a new class of nootropic drugs with an in vivo pharmacological profile very similar to that of piracetam, showing much higher potency with respect to the reference compound. Among the compounds studied, 13 (DM235) shows outstanding potency, being active at a dose of 0.001 mg kg(-1) sc.

Mono- and disubstituted-3,8-diazabicyclo[3.2.1]octane derivatives as analgesics structurally related to epibatidine: synthesis, activity, and modeling.

A series of 3,8-diazabicyclo[3.2.1]octanes substituted either at the 3 position (compounds 1) or at the 8 position (compounds 2) by a chlorinated heteroaryl ring were synthesized, as potential analogues of the potent natural analgesic epibatidine. When tested in the hot plate assay, the majority of the compounds showed significant effects, the most interesting being the 3-(6-chloro-3-pyridazinyl)-3,8-diazabicyclo[3.2.1]octane (1a). At a subcutaneous dose of 1 mg/kg, 1a induced a significant increase in the pain threshold, its action lasting for about 45 min. 1a also demonstrated good protection at a dose of 5 mg/kg in the mouse abdominal constriction test, while at 20 mg/kg it completely prevented the constrictions in the animals. Administration of naloxone (1 mg/kg i.p.) did not antagonize its antinociception while mecamylamine (2 mg/kg i.p.) did, thus suggesting the involvement of the nicotinic system in its action. Binding studies confirmed high affinity for the alpha 4 beta 2 nAChR subtype (Ki = 4.1 +/- 0.21 nM). nAChR functional activity studies on three different cell lines showed that 1a was devoid of any activity at the neuromuscular junction. Finally, due to the analogy in their pharmacological profile with that of epibatidine, compounds were compared from a structural and conformational point of view through theoretical calculations and high-field 1H NMR spectroscopy. Results indicate that all of them present one conformation similar to that of epibatidine.

Antihistamine antinociception is mediated by Gi-protein activation.

The effect of the i.c.v. administration of antisense oligodeoxynucleotides directed against the alpha subunit of different Gi-proteins (anti-Gi alpha(1), anti-Gi alpha(2), anti-Gi alpha(3)) on the antinociception induced by the H(1)-antihistamines was evaluated in the mouse hot-plate test. The administration of diphenhydramine (20 mg kg(-1) s.c.), pyrilamine (15 mg kg(-1) s.c.) and promethazine (6 mg kg(-1) s.c.) produced an increase of the pain threshold which peaked 15 min after injection. Pretreatment with anti-Gi alpha(1) (12.5 microg per mouse i.c.v.), anti-Gi alpha(2) (25 microg per mouse i.c.v.) and anti-Gi alpha(3) (25 microg per mouse i.c.v.), administered 24 and 18 h before test, prevented the antihistamine-induced antinociception. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest an important role played by the Gi-protein pathway in the transduction mechanism involved in the enhancement of the pain threshold produced by H(1)-antihistamines.

Alpha-2 agonists induce amnesia through activation of the Gi-protein signalling pathway.

The post-receptorial mechanism of the amnesic action of the alpha2-agonists clonidine and guanabenz was investigated in the mouse passive avoidance test. Animals were i.c.v. injected with pertussis toxin (PTX) or with antisense oligonucleotides, complementary to the sequence of the alpha-subunit mRNA of Gi1, Gi2, Gi3, Go1 and Go2 proteins. The administration of PTX (0.25 microg per mouse i.c.v.) reversed the amnesia induced by both alpha2-agonists. Similarly, anti-Gialpha1 (6.25-12.5 microg per mouse i.c.v.), anti-Gialpha3 (3.12-12.5 microg per mouse i.c.v.), anti-Goalpha1 (12.5-25 microg per mouse i.c.v.) antagonised the detrimental effect induced by clonidine and guanabenz. By contrast, pretreatment with anti-Gialpha2 (3.12-25 microg per mouse i.c.v.) and anti-Goalpha2 (12.5-25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by the alpha2-agonists. At the highest effective doses, none of the compounds used impaired motor coordination (rota rod test), nor modified spontaneous motility and inspection activity, (hole board test). These results indicate the involvement of Gi1, Gi3, and Go1, but not Gi2 and Go2, protein subtypes in the transduction mechanism responsible for the induction of amnesia by clonidine and guanabenz.

Diphenhydramine-induced amnesia is mediated by Gi-protein activation.

The effect of the i.c.v. administration of antisense oligodeoxynucleotides directed against the alpha subunit of different Gi-proteins (anti-Gialpha(1), anti-Gialpha(2), anti-Gialpha(3), anti-Goalpha(1), anti-Goalpha(2)) on the amnesia induced by the H(1)-antihistamine diphenhydramine (20 mg kg(-1) s.c.) was evaluated in the mouse passive avoidance test. Pretreatment with anti-Gialpha(1) (12.5-25 microg per mouse i.c.v.) and anti-Gialpha(2) (25 microg per mouse i.c.v.), administered 24 and 18 h before test, prevented antihistamine-induced amnesia. By contrast, pretreatment with an anti-Gialpha(3) (25 microg per mouse i.c.v.), anti-Goalpha(1) (25 microg per mouse i.c.v.) and anti-Goalpha(2) (25 microg per mouse i.c.v.) did not modify the detrimental effect induced by diphenhydramine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by the Gi(1)- and Gi(2)-protein pathway in the transduction mechanism involved in the impairment of memory processes produced by the H(1)-antihistamine diphenhydramine.

Loss of muscarinic antinociception by antisense inhibition of M(1) receptors.

The effect on cholinergic analgesia of inactivation of the M(1) 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-M(1) 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 microg 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-M(1) 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 M(1) 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 M(1) muscarinic receptor subtype is fundamental to induce central cholinergic analgesia in mice.

Differential prevention of morphine amnesia by antisense oligodeoxynucleotides directed against various Gi-protein alpha subunits.

The effect of the i.c.v. administration of pertussis toxin (PTX) and antisense oligodeoxynucleotide directed against the alpha subunit of different Gi-proteins (anti-Gialpha1, anti-Gialpha2, anti-Gialpha3) on amnesia induced by morphine was evaluated in the mouse passive avoidance test. The administration of morphine (6 - 10 mg kg(-1) i.p.) immediately after the training session produced amnesia that was prevented by PTX (0.25 microg per mouse i.c.v.) administered 7 days before the passive avoidance test. Anti-Gialpha1 (6.25 microg per mouse i.c.v.) and anti-Gialpha3 (12.5 microg per mouse i.c.v.), administered 18 and 24 h before the training session, prevented the morphine amnesia. By contrast, pretreatment with anti-Gialpha2 (3.12 - 25 microg per mouse i.c.v.) never modified the impairment of memory processes induced by morphine. At the highest effective doses, none of the compounds used impaired motor coordination, as revealed by the rota rod test, nor modified spontaneous motility and inspection activity, as revealed by the hole board test. These results suggest the important role played by Gi1 and Gi3 protein subtypes in the transduction mechanism involved in the impairment of memory processes produced by morphine.