Antibiotic-induced microbiota perturbation causes gut endocannabinoidome changes, hippocampal neuroglial reorganization and depression in mice.

The microbiota-gut-brain axis (MGBA) regulates the reciprocal interaction between chronic inflammatory bowel and psychiatric disorders. This interaction involves multiple pathways that are highly debated. We examined the behavioural, biochemical and electrophysiological alterations, as well as gut microbiota composition in a model of antibiotic-induced experimental dysbiosis. Inflammation of the small intestine was also assessed. Mice were exposed to a mixture of antimicrobials for 2weeks. Afterwards, they received Lactobacillus casei DG (LCDG) or a vehicle for up to 7days via oral gavage. Perturbation of microbiota was accompanied by a general inflammatory state and alteration of some endocannabinoidome members in the gut. Behavioural changes, including increased immobility in the tail suspension test and reduced social recognition were observed, and were associated with altered BDNF/TrkB signalling, TRPV1 phosphorylation and neuronal firing in the hippocampus. Moreover, morphological rearrangements of non-neuronal cells in brain areas controlling emotional behaviour were detected. Subsequent probiotic administration, compared with vehicle, counteracted most of these gut inflammatory, behavioural, biochemical and functional alterations. Interestingly, levels of Lachnospiraceae were found to significantly correlate with the behavioural changes observed in dysbiotic mice. Our findings clarify some of the biomolecular and functional modifications leading to the development of affective disorders associated with gut microbiota alterations.

Genetic variability of two Italian indigenous chicken breeds inferred from microsatellite marker analysis.

The objective of this study was to determine the genetic structure and variability of Bionda Piemontese and Bianca di Saluzzo (Piedmont, Northwest Italy) using an international set of microsatellite loci (AVIANDIV-FAO). Differences compared with commercial lines and other Italian breeds were verified to justify the implementation of conservation programmes. Flock contribution to genetic variability was assessed following the approach implemented in the MolKin software. Comparison was performed using the fixation index and the Reynolds genetic distance. The most likely number of different populations was estimated using the clustering procedure implemented in STRUCTURE. The molecular information suggests that management practices could have prevented random mating and produced inbreeding and heterogeneity across flocks. In this respect, Bionda and Bianca show substructuring and are more similar to British breeds than other continental European breeds. Bionda and Bianca fit into the European breeds provided with the highest number of alleles and expected heterozygosity. There is a clear distinction between the Piedmont breeds and the other populations. The Piedmont poultry differ from both commercial lines and other Italian breeds and retain a high level of genetic variability. As for other indigenous breeds, Bionda and Bianca could make an original contribution to the industry in the future. A collective planned approach to restoration is essential, because the flocks are managed with poor regulation. Enhancing connection between breeders with an efficient replacement interchange and mating plan is the right way of controlling inbreeding, preventing substructuring and increasing variability within the flocks.

PC1, a non-peptide PKR1-preferring antagonist, reduces pain behavior and spinal neuronal sensitization in neuropathic mice.

Peripheral neuropathy is characterized by abnormal pain responses triggered by the release of several mediators and neuronal hyperexcitability at the spinal cord level. Emerging evidence indicates that the enhanced activity of dorsal horn neurons requires communication with glia and microglia, cells that are physiologically involved in neuronal wellbeing. Prokineticins (PKs), which include PK1 and PK2, represent a novel family of chemokines characterized by a unique structural motif comprising five disulfide bonds. They are expressed in the peripheral and central nervous system. PKs bind two G protein coupled receptors, PKR1 and PKR2, and participate in the regulation of several biological processes, including pain sensation. This study aimed to investigate the anti-nociceptive effect of PC1, a non-peptide PKR1-preferring antagonist, in a mouse model of neuropathic pain. To do this, we assessed the activity of spinal cord nociceptive neurons as well as astrocyte and microglia phenotypes after repeated administration of PC1 in vivo. PC1 treatment strongly delayed the development of thermal hyperalgesia and tactile and mechanical allodynia. It also reduced spinal microglial and glial activation 8 days post injury in spared nerve injury (SNI) mice. Neuropathic mice showed an increased level of PK2 protein in the spinal cord, mostly in astrocytes. PC1 treatment completely reversed the increased responsiveness to mechanical stimuli, the decreased threshold of neuronal activation, and the increased spontaneous activity that were observed in nociceptive specific (NS) neurons of SNI mice.

Association between cannabinoid receptor type 2 Q63R variant and oligo/polyarticular juvenile idiopathic arthritis.

OBJECTIVES: To investigate whether the functional variant Q63R of the cannabinoid 2 (CB2) receptor is associated with susceptibility to oligo/poly-articular juvenile idiopathic arthritis (JIA) and with its clinical features. METHOD: A total of 171 Italian children with oligoarticular/rheumatoid factor negative poly-articular JIA and 600 healthy controls were enrolled in the study and genotyped. RESULTS: A significant difference in genotype distribution of the CB2 Q63R variant (CNR2 rs35761398) between oligo/poly-articular JIA patients and controls was found (p = 0.001). The R63 variant was associated with increased rates of relapse (p = 0.0001). CONCLUSIONS: This study indicates that the CB2 receptor contributes to susceptibility to oligo/polyarticular JIA and to the severity of its clinical course.

The A1 adenosine receptor as a new player in microglia physiology.

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

NAbiximols Clinical Translation To the treatment of Pain and Agitation In Severe Dementia (NACTOPAISD): Clinical trial protocol.

Up to 80 % nursing home residents with dementia experiences chronic pain. Contextually, 97 % presents fluctuant neuropsychiatric symptoms (NPS). Among the most challenging is agitation, connected with undertreated pain and managed through neuroleptics doubling death risk. Evidence is accumulating in favor of the involvement of the endocannabinoid system in nociception and NPS. This double-blind, placebo-controlled, randomized trial (NAbiximols Clinical Translation To the treatment of Pain and Agitation In Severe Dementia [NACTOPAISD]) aims at investigating efficacy and safety of oral spray nabiximols, containing Δ9-tetrahydrocannabinol and cannabidiol (Sativex®), for pain and agitation treatment in severe dementia patients (Mini-Mental State Examination ≤ 12) over 65. The coprimary endpoints are efficacy on pain and agitation, assessed through the recently validated Italian Mobilization-Observation-Behavior-Intensity-Dementia and the Cohen-Mansfield Agitation Inventory. The secondary endpoint is the evaluation of efficacy duration after wash-out and the assessment of quality of life through the DEMQOL. Any adverse events will be reported. The results undergo statistical analysis plan. NACTOPAISD might provide rationale for a translational safer pain and agitation treatment in severe dementia. It is approved by Calabria Region Ethics Committee and follows the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) and the Consolidated Standards of Reporting Trials (CONSORT) statements.

Is there a rational basis for cannabinoids research and development in ocular pain therapy? A systematic review of preclinical evidence.

BACKGROUND: Purpose of the present systematic review is to investigate preclinical evidence in favor of the working hypothesis of efficacy of cannabinoids in ocular pain treatment. METHODS: Literature search includes the most relevant repositories for medical scientific literature from inception until November, 24 2021. Data collection and selection of retrieved records adhere to PRISMA criteria. RESULTS: In agreement with a priori established protocol the search retrieved 2471 records leaving 479 results after duplicates removal. Eleven records result from title and abstract screening to meet the inclusion criteria; only 4 results are eligible for inclusion in the qualitative synthesis impeding meta-analysis. The qualitative analysis highlights the antinociceptive and anti-inflammatory efficacy of Δ8-tetrahydrocannabinol, cannabidiol and its derivative HU-308 and of new racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229. Moreover, CB2R agonists RO6871304 and RO6871085 and CB2R ligand HU910 provide evidence of anti-inflammatory efficacy. CB2 agonist HU308 reduces of 241% uveitis-induced leukocyte adhesion and changes lipidome profile. Methodological and design issues raise concern of risk of bias and the amount of studies is too small for generalization. Furthermore, the ocular pain model used can resemble only inflammatory but not neuropathic pain. CONCLUSIONS: The role of the endocannabinoid system in ocular pain is underinvestigated, since only two studies assessing the effects of cannabinoid receptors modulators on pain behavior and other two on pain-related inflammatory processes are found. Preclinical studies investigating the efficacy of cannabinoids in ocular inflammatory and neuropathic pain models are needed to pave the way for clinical translation.

2-pentadecyl-2-oxazoline prevents cognitive and social behaviour impairments in the Amyloid ß-induced Alzheimer-like mice model: Bring the α2 adrenergic receptor back into play.

The 2-pentadecyl-2-oxazoline (PEA-OXA) is a natural compound with protective action in neuro-inflammation. We have previously shown that PEA-OXA behaves as an α2 adrenergic receptor (α2AR) antagonist and a putative protean agonist on histamine H3 receptors. Recently, neuroinflammation and monoaminergic neurotransmission dysfunction has drawn particular attention in Alzheimer Disease (AD) pathophysiology. In this context, the objective of this study was to investigate the effects of the dual-acting PEA-OXA in an AD-like model in mice. A combined computational and experimental approach was used to evaluate the ability of PEA-OXA to bind α2A-AR subtype, and to investigate the effects of PEA-OXA treatment on neuropathological (behavioural and functional) effects induced by soluble Amyloid ß 1-42 (sAß1-42) intracerebroventricular injection. Computational analysis revealed the PEA-OXA ability to bind the α2A-AR, a pharmacological target for AD, in two alternative poses, one overlapping the Na+ binding site. In vivo studies indicated that chronic treatment with PEA-OXA (10 mg/kg, os) restored the cognitive (discriminative and spatial memory) deficits and social impairments induced by sAß injection. Consistently, electrophysiological analysis showed a recovery of the long-term potentiation in the hippocampus (Lateral Entorhinal Cortex-Dentate Gyrus pathway), while neuroinflammation, i.e., increased pro-inflammatory cytokines levels and microglia cells density were reduced. These data provide the basis for further investigation of the pro-cognitive aptitude of PEA-OXA by proposing it as an adjuvant in the treatment in AD, for which the available pharmacological approaches remain unsatisfactory. Moreover, this study offers new future direction in research investigating the role of α2AR in neuropsychiatric illness and therapies.

Cannabidiol in the prelimbic cortex modulates the comorbid condition between the chronic neuropathic pain and depression-like behaviour in rats: The role of medial prefrontal cortex 5-HT1A and CB1 receptors.

The prelimbic division (PrL) of the medial prefrontal cortex (mPFC) is a cerebral division that is putatively implicated in the chronic pain and depression. We investigated the activity of PrL cortex neurons in Wistar rats that underwent chronic constriction injury (CCI) of sciatic nerve and were further subjected to the forced swimming (FS) test and mechanical allodynia (by von Frey test). The effect of blockade of synapses with cobalt chloride (CoCl2), and the treatment of the PrL cortex with cannabidiol (CBD), the CB1 receptor antagonist AM251 and the 5-HT1A receptor antagonist WAY-100635 were also investigated. Our results showed that CoCl2 decreased the time spent immobile during the FS test but did not alter mechanical allodynia. CBD (at 15, 30 and 60 nmol) in the PrL cortex also decreased the frequency and duration of immobility; however, only the dose of 30 nmol of CBD attenuated mechanical allodynia in rats with chronic NP. AM251 and WAY-100635 in the PrL cortex attenuated the antidepressive and analgesic effect caused by CBD but did not alter the immobility and the mechanical allodynia when administered alone. These data show that the PrL cortex is part of the neural substrate underlying the comorbidity between NP and depression. Also, the previous blockade of CB1 cannabinoid receptors and 5-HT1A serotonergic receptors in the PrL cortex attenuated the antidepressive and analgesics effect of the CBD. They also suggest that CBD could be a potential medicine for the treatment of depressive and pain symptoms in patients with chronic NP/depression comorbidity.

1-(2',4'-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide, a novel CB2 agonist, alleviates neuropathic pain through functional microglial changes in mice.

Neuropathic pain is a devastating neurological disease that seriously affects quality of life in patients. The mechanisms leading to the development and maintenance of neuropathic pain are still poorly understood. However, recent evidence points towards a role of spinal microglia in the modulation of neuronal mechanisms. In this context, cannabinoids are thought to modulate synaptic plasticity as well as glial functions. Here, we have investigated the effect of chronic treatment with a selective agonist of cannabinoid type 2 receptor (CB2), 1-(2',4'-dichlorophenyl)-6-methyl-N-cyclohexylamine-1,4-dihydroindeno[1,2-c]pyrazole-3 carboxamide (NESS400), on pain thresholds in the spared nerve injury (SNI) model in the mouse and on the distribution and activation of spinal microglia. Repeated treatment with NESS400 (4 mg/kg) significantly alleviated neuropathic mechanical allodynia and thermal hyperalgesia. In the dorsal horn (L4-L6) of neuropathic mice microglia activation (quantification of the length of microglial processes) and astrocytosis were associated with CB2 receptor over-expression on both cell types. Treatment with NESS400 significantly reduced the number of hypertrophic microglia while leaving microglial cell number unaffected and reduced astrogliosis. Moreover, prolonged administration of NESS400 reduced mRNA expression of pro-inflammatory markers and enhanced anti-inflammatory marker gene expression in dorsal horn extracts. In conclusion, we show that selective CB2 receptor stimulation prevents thermal hyperalgesia, alleviates mechanical allodynia and facilitates the proliferation of anti-inflammatory microglial phenotype in the ipsilateral dorsal horn of the spinal cord in SNI mice.

The Cold Case of Metabotropic Glutamate Receptor 6: Unjust Detention in the Retina?

It is a common opinion that metabotropic glutamate receptor subtype 6 (mGluR6) is expressed exclusively in the retina, and in particular in the dendrites of ON-bipolar cells. Glutamate released in darkness from photoreceptors activates mGluR6, which is negatively associated with a membrane non-selective cation channel, the transient receptor potential melanoma-related 1, TRPM1, resulting in cell hyperpolarization. The evidence that mGluR6 is expressed not only in the retina but also in other tissues and cell populations has accumulated over time. The expression of mGluR6 has been identified in microglia, bone marrow stromal and prostate cancer cells, B lymphocytes, melanocytes and keratinocytes and non-neural tissues such as testis, kidney, cornea, conjunctiva, and eyelid. The receptor also appears to be expressed in brain areas, such as the hypothalamus, cortex, hippocampus, nucleus of tractus solitarius, superior colliculus, axons of the corpus callosum and accessory olfactory bulb. The pharmacological activation of mGluR6 in the hippocampus produced an anxiolytic-like effect and in the periaqueductal gray analgesic potential. This review aims to collect all the evidence on the expression and functioning of mGluR6 outside the retina that has been accumulated over the years for a broader view of the potential of the receptor whose retinal confinement appears understimated.

Is ITS-2 rDNA suitable marker for genetic characterization of Sarcoptes mites from different wild animals in different geographic areas?

The present study examined the relationship among individual Sarcoptes scabiei mites from 13 wild mammalian populations belonging to nine species in four European countries using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) as genetic marker. The ITS-2 plus primer flanking 5.8S and 28S rDNA (ITS-2+) was amplified from individual mites by polymerase chain reaction (PCR) and the amplicons were sequenced directly. A total of 148 ITS-2+ sequences of 404bp in length were obtained and 67 variable sites were identified (16.59%). UPGMA analyses did not show any geographical or host-specific clustering, and a similar outcome was obtained using population pairwise Fst statistics. These results demonstrated that ITS-2 rDNA does not appear to be suitable for examining genetic diversity among mite populations.

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy.

AIMS: To determine the variability of the prion protein gene (PRNP) in goats from Northern and Southern Italy. METHODS AND RESULTS: Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis. CONCLUSIONS: Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Effects of (S)-3,4-DCPG, an mGlu8 receptor agonist, on inflammatory and neuropathic pain in mice.

In this study, the effect of (S)-3,4-dicarboxyphenylglycine (DCPG), a selective mGlu8 receptor agonist, has been investigated in inflammatory and neuropathic pain models in order to elucidate the role of mGlu8 receptor in modulating pain perception. Inflammatory pain was induced by the peripheral injection of formalin or carrageenan in awake mice. Systemic administration of (S)-3,4-DCPG, performed 15 min before formalin, decreased both early and delayed nociceptive responses of the formalin test. When this treatment was carried out 15 min after the peripheral injection of formalin it still reduced the late hyperalgesic phase. Similarly, systemic (S)-3,4-DCPG reduced carrageenan-induced thermal hyperalgesia and mechanical allodynia when administered 15 min before carrageenan, but no effect on pain behaviour was observed when (S)-3,4-DCPG was given after the development of carrageenan-induced inflammatory pain. When microinjected into the lateral PAG (RS)-alpha-methylserine-O-phoshate (MSOP), a group III receptor antagonist, antagonised the analgesic effect induced by systemic administration of (S)-3,4-DCPG in both of the inflammatory pain models. Intra-lateral PAG (S)-3,4-DCPG reduced pain behaviour when administered 10 min before formalin or carrageenan; both the effects were blocked by intra-lateral PAG MSOP. (S)-3,4-DCPG was ineffective in alleviating thermal hyperalgesia and mechanical allodynia 7 days after the chronic constriction injury of the sciatic nerve, whereas it proved effective 3 days after surgery. Taken together these results suggest that stimulation of mGlu8 receptors relieve formalin and carrageenan-induced hyperalgesia in inflammatory pain, whereas it would seem less effective in established inflammatory or neuropathic pain.

Analgesic actions of N-arachidonoyl-serotonin, a fatty acid amide hydrolase inhibitor with antagonistic activity at vanilloid TRPV1 receptors.

BACKGROUND AND PURPOSE: N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia. EXPERIMENTAL APPROACH: AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured. KEY RESULTS: AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM251, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone. CONCLUSIONS AND IMPLICATIONS: Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.

Analysis of the sheep MUC1 gene: structure of the repetitive region and polymorphism.

An investigation was undertaken with the aim of studying the repetitive region of the MUC1 gene and analyzing its polymorphisms in some Italian sheep breeds. Two primers previously used for the goat MUC1 gene analyses allowed for the amplification of 4 different alleles. The sequence analysis showed that the repetitive region of the sheep MUC1 gene is an array of 60-bp repeats, in accordance with the information reported in humans, cattle, and goats. The polypeptide sequence encoded by the consensus repeat was very similar to the corresponding sequences of goats and cattle. The average homology of all repeated units was 82%; when the repeats were compared with the derived consensus repeat, homology dropped to 78%. The repeats were not all perfectly conserved, but the sequence homology was nevertheless clearly sufficient to preserve the mechanism giving rise to the variable-number tandem-repeat polymorphism. In spite of their reduced sequence homology, the sheep repeats shared a high number of potential glycosylation sites. The conservation of the exact number and position of glycosylation sites did not seem to be very important for the purpose of functional integrity, but glycosylation appeared to be conserved as a bulk property. Analysis of the polymorphism in 6 Italian breeds showed that the sheep repetitive region seemed to be less variable and smaller in size than the repetitive region of the goat. The findings of this study suggest that ruminants can be a useful model to study the mechanisms by which the variation in the repeat number and the extracellular domain size can modulate the effectiveness of MUC1 as a cell-surface shield.

Palmitoylethanolamide induces microglia changes associated with increased migration and phagocytic activity: involvement of the CB2 receptor.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Comparative FISH mapping of mucin 1, transmembrane (MUC1) among cattle, river buffalo, sheep and goat chromosomes: comparison between bovine chromosome 3 and human chromosome 1.

Four bovine BAC clones (0494F01, 0069D07, 0060B06, and 0306A12) containing MUC1, as confirmed by mapping MUC1 on a RH3000 radiation hybrid panel, were hybridised on R-banded chromosomes of cattle (BTA), river buffalo (BBU), sheep (OAR) and goat (CHI). MUC1 was FISH-mapped on BTA3q13, BBU6q13, OAR1p13 and CHI3q13 and both chromosomes and chromosome bands were homoeologous confirming the high degree of chromosome homoeologies among bovids and adding more information on the pericentromeric regions of these species' chromosomes. Indeed, MUC1 was more precisely assigned to BTA3 and assigned for the first time to BBU6, OAR1p and CHI3. Moreover, detailed and improved cytogenetic maps of BTA3, CHI3, OAR1p and BBU6 are shown and compared with HSA1.

In vitro and in vivo pharmacology of synthetic olivetol- or resorcinol-derived cannabinoid receptor ligands.

BACKGROUND AND PURPOSE: We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. EXPERIMENTAL APPROACH: The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. KEY RESULTS: CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CONCLUSIONS AND IMPLICATIONS: CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

Metabotropic glutamate and cannabinoid receptor crosstalk in periaqueductal grey pain processing.

Metabotropic glutamate (mGlu) and cannabinoid receptors are G-protein coupled receptors which have shown synaptic co-operation through small lipid messengers in the central nervous system (CNS). A functional interaction between these two receptor families could have a relevant potential in the treatment of CNS disorders, including chronic pain. Indeed, both mGlu and cannabinoid receptors play a crucial role in the neurobiology of pain and their simultaneous manipulation could lead to novel strategies in pain management. In particular, as both mGlu and cannabinoid receptors have been found in the periaqueductal gray (PAG), a crucial station in the pain modulatory system, these receptors could be a substrate for producing analgesia at this level. In this review we aim to briefly illustrate the role of mGlu and can-nabinoid receptors in controlling nociceptive processes, some points of convergence, and their functional interaction in pain processing. Further insights into this functional linkage between the mGlu and cannabinoid receptors could pave the way to a new strategy for pain relief, such as a drug cocktail acting on cannabinoid/metabotropic glutamate receptors.