Biomedical subjects

Luca Pani

Publications and source records attributed to Luca Pani.

31 records · Page 2Linked to original sources

PubMed2003

Characterization of wild-type (R100R) and mutated (Q100Q) GABAA alpha 6 subunit in Sardinian alcohol non-preferring rats (sNP).

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA(A) receptor alpha(6) subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using [3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of [3H]Ro 15-4513 for GABA(A) receptors in QQ with respect to RR rats (K(d) values 4.0+/-0.67 and 6.24+/-0.95 nM, respectively), with similar B(max) values (3.5+/-0.25 and 3.9+/-0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K(i1) values for QQ and RR (3.6+/-0.62 and 4.9+/-0.33 nM, respectively). In QQ rats diazepam is able to completely displace [3H]Ro 15-4513 (K(i2)=1.48+/-0.27 microM), while in RR rats the diazepam sensitive sites are still present (K(i2)>10 microM). The basal mRNA and protein expression level of the alpha(6) subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA(A) alpha(6) subunit and its involvement in determining alcohol related behavior.

Alcohol Drinking↗

PubMed2003

Synthesis and characterization of NESS 0327: a novel putative antagonist of the CB1 cannabinoid receptor.

The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo [6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS 0327) was synthesized and evaluated for binding affinity toward cannabinoid CB1 and CB2 receptor. NESS 0327 exhibited a stronger selectivity for CB1 receptor compared with N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A), showing a much higher affinity for CB1 receptor (Ki = 350 +/- 5 fM and 1.8 +/- 0.075 nM, respectively) and a higher affinity for the CB2 receptor (Ki = 21 +/- 0.5 nM and 514 +/- 30 nM, respectively). Affinity ratios demonstrated that NESS 0327 was more than 60,000-fold selective for the CB1 receptor, whereas SR 141716A only 285-fold. NESS 0327 alone did not produce concentration-dependent stimulation of guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding in rat cerebella membranes. Conversely, NESS 0327 antagonized [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (WIN 55,212-2)-stimulated [35S]GTPgammaS binding. In functional assay, NESS 0327 antagonized the inhibitory effects of WIN 55,212-2 on electrically evoked contractions in mouse isolated vas deferens preparations with pA2 value of 12.46 +/- 0.23. In vivo studies indicated that NESS 0327 antagonized the antinociceptive effect produced by WIN 55,212-2 (2 mg/kg s.c.) in both tail-flick (ID50 = 0.042 +/- 0.01 mg/kg i.p.) and hot-plate test (ID50 = 0.018 +/- 0.006 mg/kg i.p.). These results indicated that NESS 0327 is a novel cannabinoid antagonist with high selectivity for the cannabinoid CB1 receptor.

Animals↗

PubMed2003

Co-release of noradrenaline and dopamine from noradrenergic neurons in the cerebral cortex induced by clozapine, the prototype atypical antipsychotic.

RATIONALE: Clozapine has been shown to increase extracellular dopamine (DA) and noradrenaline (NA) in the medial prefrontal cortex (mPFC). A recent study of ours suggested that extracellular DA in the PFC originates not only from dopaminergic but also from noradrenergic terminals, its release being controlled by alpha(2)-adrenoceptors. OBJECTIVES: Since clozapine binds to alpha(2)-adrenoceptors, the possibility that it might co-release DA and NA was studied. METHODS: By means of microdialysis coupled to HPLC with electrochemical detection, the effect of clozapine on extracellular DA and NA in the mPFC, densely innervated by DA and NA, was compared to that in the occipital cortex, equally innervated by NA but receiving few DA projections. RESULTS: Extracellular NA was found to be the same in the two cortices, consistent with homogeneous NA innervation. On the other hand, extracellular DA in the occipital cortex was only 29% lower than in the mPFC, in spite of the scarce dopaminergic innervation in the occipital cortex. Clozapine (10 mg/kg IP) increased extracellular DA and NA not only in the mPFC (by about 320% and 290%, respectively) but also in the occipital cortex (by 560% and 230%, respectively). Administration of the alpha(2)-agonist clonidine (0.15 mg/kg) reversed the effect of clozapine in both cortices, while the D(2)-agonist quinpirole (0.1 mg/kg IP) was ineffective. CONCLUSIONS: The results suggest that clozapine, by inhibiting alpha(2)-adrenoceptors, co-releases DA and NA from noradrenergic terminals in the occipital cortex and that the same mechanism might be responsible for the concomitant increase of the two monoamines in the mPFC.

Adrenergic alpha-Agonists↗

PubMed2003

Molecular characterization of new polymorphisms at the beta2, alpha1, gamma2 GABA(A) receptor subunit genes associated to a rat nonpreferring ethanol phenotype.

Recent preclinical and clinical studies have indicated a possible involvement of the genes encoding for the GABA(A) receptor subunits alpha6, beta2, alpha1 and gamma2 in the genetic susceptibility to alcohol abuse. We have recently found an (R) to (Q) mutation in codon 100 of the alpha6 GABA(A) subunit, that segregated in a rat line selectively bred for its voluntary ethanol aversion, Sardinian alcohol nonpreferring (sNP), but not in their Sardinian alcohol preferring (sP) counterpart, selected for its ethanol preference. In the present study the molecular composition of other GABA(A) subunits (beta2, alpha1 and gamma2) were analyzed in order to further investigate the involvement of the GABA(A) receptors in the genetic predisposition to voluntary alcohol intake. Automated sequencing analysis indicated the presence of six new silent substitutions (289 T-->C in the beta2 gene; 115 G-->A in the alpha1 gene; 157 G-->A, 174 C-->T, 347 A-->G and 385 A-->T in the gamma2 gene), in sNP but not in sP rats. These polymorphisms were linked to the alpha6 R100Q mutation previously described in sNP rats. The strict association between the alpha6 point mutation and the new polymorphisms found in the beta2, alpha1 and gamma2 genes, demonstrate that such genes belong to the same cluster and are inherited together in the rat. These results sustain the synteny for these clusters between the rodent and human genomes, and suggest that mutated GABA(A) beta2, alpha6, alpha1 and gamma2 subunit genes might contribute to the expression of an ethanol nonpreferring phenotype in a rat line that voluntarily avoids alcoholic solutions.

Alcohol-Induced Disorders, Nervous System↗

PubMed2003

Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.

Recently, the gastrointestinal pharmacology of cannabinoid CB(1) receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB(1) receptor protein in the mouse gastroenteric tract. The cannabinoid CB(1) receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB(1) receptor protein. The immunohistochemical study showed intense cannabinoid CB(1) receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB(1) receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 microg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB(1) antagonist SR 141716A.

Animals↗

PubMed2003

In vitro evidence for the presence of [3H]-haloperidol uptake in rat brain.

1 The neuroleptic [(3)H]-haloperidol (HP) was taken up in synaptosomes prepared from rat brain, in a temperature-, sodium ion-, and energy-dependent process. 2 The highest concentration of uptake sites (V(max)=2.37 pmol mg(-1) protein min(-1)) was in the striatum with the other brain areas displaying lower (by 50-70%) values. 3 The affinity values (K(m) approximately equal to 40 nM) were similar in all brain areas considered. 4 The pharmacological characterization did not indicate a well-defined group of inhibitors, which suggested that HP might not use a transporter for recognized neurotransmitters. 5 The HP metabolites tested, including HPTP, were competitive inhibitors of [(3)H]-HP uptake, an indirect indication that they may actively enter the striatal nerve endings through the same carrier. 6 Since the uptake process was partially affected by the incubation of [(3)H]-HP in the presence of several antagonists of HP-transforming cytochrome P450 isoforms, the binding of HP at some enzyme sites inside the synaptosome cannot be excluded. 7 In conclusion, the present results suggest that HP may be actively transported in the rat brain.

Animals↗

PubMed2002

Reduced DAT- and DBH-immunostaining in the limbic system of Sardinian alcohol-preferring rats.

We have recently shown that tyrosine-hydroxylase immunostaining (TH-IM) is selectively decreased in the cingulate cortex and in the shell of the nucleus accumbens (nAcc) of Sardinian alcohol-preferring rats (sP) when compared with Sardinian alcohol-non preferring (sNP) and Wistar (W) rats. Since these regions contain both dopamine and noradrenaline (NA) fibers, clarification of the dopaminergic and noradrenergic contribution to the decreased TH-immunoreactivity was needed. To this aim, we carried out the present immunohistochemistry study using two antibodies raised against dopamine beta-hydroxylase (DBH), the enzyme responsible for the conversion of dopamine into noradrenaline, and against the dopamine transporter (DAT), as markers for noradrenergic and dopaminergic fibers, respectively. The results show that DBH-immunostaining (DBH-IM) and DAT-immunostaining (DAT-IM) were both lower in the cingulate cortex of the sP rats with respect to sNP and W rats. In the shell of the nAcc a reduced DAT-IM in sP rats was found, while the DBH-IM did not differ between the three lines of rats. The analysis of the cell-body area of noradrenergic neurons in the locus coeruleus, revealed no differences between sP, sNP and W rats. These results indicate a selective reduction of the terminal innervation in the mesocorticolimbic dopamine and NA systems in sP rats. This genetically-determined difference may be involved in the opposite alcohol preference and consumption of sP and sNP rats.

Alcohol Drinking↗

PubMed2002

Effect of the amisulpride isomers on rat prolactinemia.

The effects on rat serum prolactin level of the two isomers constituting the racemic form of amisulpride were compared. (S-)-amisulpride induced hyperprolactinemia at lower doses (ED(50) = 0.09 +/- 0.01 mg/kg) than racemic- (ED(50) = 0.24 +/- 0.03 mg/kg) and (R+)-amisulpride (ED(50) = 4.13 +/- 0.05 mg/kg), in accord with their affinities for pituitary dopamine D(2) receptor (K(i) = 3.8 +/- 0.2, 6.4 +/- 0.2 and 143.3 +/- 2.3 nM, respectively). At doses twice the ED(50), (S-)-amisulpride produced a maximal increase in prolactin level similar to that of the racemic form (403 +/- 21% and 425 +/- 15%, respectively), but higher than that of (R+)-amisulpride (198 +/- 8%). These results suggest that the hyperprolactinemia induced by the racemic-amisulpride is mostly due to its (S-)-isomer.

Amisulpride↗

PubMed2002

Carmoxirole is able to reduce amisulpride-induced hyperprolactinemia without affecting its central effect.

Prolactin blood level and apomorphine-induced yawning were studied in rats treated with the substituted benzamide amisulpride in association with bromocriptine or carmoxirole; two dopamine D(2) receptor agonists with high or low propensity to cross the brain-blood barrier, respectively. Administration of amisulpride produced a maximum increase in rat serum prolactin level (315+/-18%) vs. vehicle-treated animals (ED(50)=0.25+/-0.017 mg/kg, s.c.). The concurrent administration of carmoxirole or bromocriptine completely reversed the hyperprolactinemia induced by amisulpride (0.5 mg/kg, s.c.) (ID(50)=14.9+/-0.8 mg/kg and 0.81+/-0.03 mg/kg, respectively). Carmoxirole (15 mg/kg, i.p.) did not affect yawning induced by apomorphine (0.08 mg/kg, s.c.) nor amisulpride (0.5 mg/kg, s.c.) blockade of apomorphine-induced yawning. Conversely, a significant increase in the number of yawns was observed when bromocriptine (0.8 mg/kg, i.p.) was associated with apomorphine in the absence or presence of amisulpride. These results suggested that a peripheral dopamine D(2) receptor agonists could be a useful tool in alleviating amisulpride-induced hyperprolactinemia without possibly affecting its central effect.

Amisulpride↗

PubMed2002

Effect of the amisulpride isomers on rat catalepsy.

The substituted benzamide amisulpride is currently administered in its racemic form. In the present study, the biochemical and cataleptogenic profiles of the two enantiomers (R+ and S-) were compared with those of the racemic mixture. Displacement binding studies showed that the (S-)-isomer possesses an higher affinity for dopamine D2-like receptor (K(i) 5.2+/-0.4 nM) compared to (R+)-amisulpride (K(i) 244+/-12 nM) and to (RS)-amisulpride (K(i) 9.8+/-0.8 nM). In contrast, (S-)-amisulpride binds the alpha(2)-receptor with an affinity (K(i) 1528+/-45 nM) lower than that of the (R+)-isomer (K(i) 375+/-34 nM) and of (RS)-amisulpride (K(i) 783+/-27 nM). The bar test was used to evaluate the catalepsy induced by each drug. (RS)-amisulpride induced catalepsy only at very high doses (>100 mg/kg, s.c.) whereas, (S-)-amisulpride produced a catalepsy at a lower dose (30 mg/kg, s.c.) and (R+)-amisulpride did not produce any catalepsy up to the dose of 75 mg/kg. Interestingly, (R+)-amisulpride reduced the catalepsy induced by (S-)-amisulpride (50 mg/kg, s.c.) or haloperidol (0.3 mg/kg, s.c.), at the doses of 50 or 30 mg/kg, respectively. These results indicate that the weak cataleptic properties of (RS)-amisulpride might partially rely on its (R+)-isomer and provide a further explanation for the atypical properties of amisulpride as an antipsychotic.

Amisulpride↗

PubMed2002

Reduced TH-immunoreactive fibers in the limbic system of Sardinian alcohol-preferring rats.

The mesolimbic dopamine (DA) system has long been known to be involved in reward behaviors. As with other substances of abuse, it has been extensively reported that ethanol influences the dopaminergic system. The present study examined whether selectively bred Sardinian-alcohol-preferring (sP) and Sardinian alcohol non-preferring rats (sNP), differ in the DA innervation in structures of the forebrain that are related to rewarding behaviors. To this aim, we performed an immunohistochemistry study with an antibody raised against tyrosine hydroxylase (TH), the rate-limited step enzyme in the biosynthesis of monoamines. The TH-positive innervation density was found to be significantly lower in the cingulate cortex and in the shell of the nucleus accumbens of the sP when compared with the sNP and unselected Wistar rats. These anatomical structures both cluster in the medial aspect of the mesolimbic system. No differences in other major DA brain regions, such as the nigro-striatal pathway were found. The analysis of cell-body area revealed no differences between sP, sNP and Wistar rats in the ventral tegmental area and substantia nigra (pars compacta and reticulata) and the density of the TH-positive fibers was not different in the caudate-putamen. These results indicate a selective reduction of terminal innervation in the medial portion of the mesocorticolimbic DA system in sP rats and suggest that the latter may consume larger amounts of ethanol, when compared with sNP rats, to compensate for the deficiency of dopamine to produce an adequate level of reward.

Alcohol Drinking↗

PubMed2002

Sub-chronic treatment with classical but not atypical antipsychotics produces morphological changes in rat nigro-striatal dopaminergic neurons directly related to "early onset" vacuous chewing.

In the present work, we investigated if an impairment of dopaminergic neurons after subchronic haloperidol treatment might be a possible physiopathologic substrate of the "early onset" vacuous chewing movements (VCMs) in rats. For this purpose, different antipsychotics were used to analyse a possible relationship between VCMs development and morphological alterations of tyrosine-hydroxylase-immunostained (TH-IM) neurons. Rats treated twice a day with haloperidol displayed a significant increase of VCMs that was both time- (2-4 weeks) and dose (0.1-1 mg/kg) dependent. Immunocytochemical analysis showed a shrinkage of TH-IM cell bodies in substantia nigra pars compacta and reticulata and a reduction of TH-immunostaining in the striatum of haloperidol treated rats with the arising of VCMs. No differences were observed in TH-IM neurons of ventral tegmental area and nucleus accumbens vs. control rats. The atypical antipsychotics risperidone (2 mg/kg, twice a day), amisulpride (20 mg/kg, twice a day) and clozapine (10 mg/kg, twice a day) did not produce any nigro-striatal morphological changes or VCMs. TH-IM nigro-striatal neuron morphological alterations and VCMs were still present after three days of withdrawal in rats treated for four weeks with haloperidol (1 mg/kg). Both the main morphological changes and the behavioural correlate disappeared after three weeks of withdrawal. These results suggest that haloperidol induces a morphological impairment of the dopaminergic nigro-striatal neurons which is directly associated with the arising, permanency and disappearance of VCMs in rats.

Amisulpride↗

PubMed2002

Evidence for functional CB1 cannabinoid receptor expressed in the rat thyroid.

OBJECTIVE: Previous reports have shown that the Delta(9)-tetrahydrocannabinol (Delta(9)TCH), the major psychoactive cannabinoid components of marijuana, is able [corrected] to inhibit thyroid hormonal activity. The aim of this study was to characterize the CB1 functional expression in the rat thyroid by a multi-methods approach. METHODS AND RESULTS: RT-PCR was used to detect the mRNA expression of the CB1 cannabinoid receptor (17.8+/-4.0% of the normalizing reference gene beta(2) microglobulin), as well as the expression of the endocannabinoid hydrolyzing enzyme, fatty acid amide hydrolase (46.9+/-4.3% of beta(2) microglobulin), in the rat thyroid gland. The CB1-encoded protein was detected in its glycosylated form (63 kDa) by Western blot, employing a polyclonal antibody, while CB1 immunohistochemical localization showed an intracellular positive staining in both follicular and parafollicular cells. In addition, a 30% decrease in serum levels of both 3,5,3' tri-iodothyronine (T(3)) and thyroxine (T(4)) was detected 4 h after the administration of the synthetic cannabinoid receptor agonist, WIN 55,212-2 (10 mg/kg i.p.). These effects were antagonized by pretreatment with the CB1 antagonist SR 141716A (3 mg/kg i.p.); thyrotrophin levels were unaffected by both treatments. CONCLUSION: These data indicate that functional CB1 receptors which are able to modulate the release of T(3) and T(4) are expressed in the rat thyroid, and suggest a possible role of cannabinoids in the regulation of rat thyroid hormonal activity.

Amidohydrolases↗