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ARL13B is a small GTPase enriched in cilia. Deletion ofArl13bin mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3.

La Commissione Europea (CE) ha inserito ad oggi 36 taxa esotici vegetali nella lista delle specie esotiche invasive di rilevanza unionale ai sensi del Regolamento (UE) n. 1143/2014 del Parlamento Europeo e del Consiglio, recante disposizioni volte a prevenire e gestire l’introduzione e la diffusione delle specie esotiche invasive. La lista delle specie di rilevanza unionale viene periodicamente aggiornata e include quelle specie che rappresentano una grave minaccia per la biodiversità, ma anche per la salute dei cittadini e le attività economiche nei territori dell’Unione Europea e che necessitano di una gestione concertata a livello comunitario. La CE vigila sullo stato di ogni taxon grazie anche a periodiche rendicontazioni da parte dei paesi dell'Unione. In vista di tali report, tra il 2020 e il 2021 è stata definita e integrata la distribuzione di queste specie in Italia.

Enforcement is critical to guarantee the effectiveness of environmental laws for nature conservation. Erroneously assuming an equivalence between the formal implementation of environmental legislation on paper and its practical enforcement in reality can result in biased conclusions with potential to ill-inform conservation actions and influence stakeholder perceptions. Here, using as an illustrative example the implementation of European sanitary regulations EC 1069/2009 and EU 142/2011 to manage livestock carcasses for wildlife conservation in Spain and Portugal, we demonstrate how the legal implementation of these regulations does not mean effective enforcement and compliance in practice. When interviewed, more Portuguese farmers declared to leave carcasses in the field without official authorization, than their Spanish counterparts, who were legally allowed to do so. This unforeseen result was further supported by GPS-tracked vultures feeding on livestock carcasses available in the Portuguese countryside, contrasting to what would be expected considering the sanitary regulations approved at each country at the time of this study. Accordingly, while agreeing with the global trend for weak enforcement and compliance with environmental legislation, our results provide additional evidence against assuming that the formal implementation on paper of environmental laws equals their real implementation on the ground. We highlight the need to systematically assess (not assume) observance of and compliance with environmental legislation and propose some ways to improve enforcement using as an example the above referred sanitary regulations. Communication-based interventions to publicize the regulations, reducing bureaucratic burden, and on-ground monitoring to assess observance and compliance have strong potential to enhance enforcement. Overlooking implementation gaps can give rise to biased interpretations on the effectiveness of these legal tools with consequences at both, the scientific and conservation arenas., PMT and JVLB were supported by a GRUPIN research grant from the Regional Government of Asturias (IDI/2021/000075). JVLB was supported by a Ramon & Cajal research contract (RYC-2015-18932) from the Spanish Ministry of Economy, Industry and Competitiveness. This research was partially financed by the LIFE Rupis project (LIFE14 NAT/PT/000855), funded by EU LIFE Programme, and by the Sentinelas project funded by Fundo Ambiental – Ministério do Ambiente e da Ação Climática.

ELMOD2 is a GTPase activating protein (GAP) with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway support a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

Shinde et al. find that activation of G protein–coupled receptors (GPCRs) inside cilia triggers formation of ubiquitin chains onto the receptors via β-arrestin and that ubiquitin chains commit the GPCRs for exit from cilia., Regulated trafficking of G protein–coupled receptors (GPCRs) controls cilium-based signaling pathways. β-Arrestin, a molecular sensor of activated GPCRs, and the BBSome, a complex of Bardet–Biedl syndrome (BBS) proteins, are required for the signal-dependent exit of ciliary GPCRs, but the functional interplay between β-arrestin and the BBSome remains elusive. Here we find that, upon activation, ciliary GPCRs become tagged with ubiquitin chains comprising K63 linkages (UbK63) in a β-arrestin–dependent manner before BBSome-mediated exit. Removal of ubiquitin acceptor residues from the somatostatin receptor 3 (SSTR3) and from the orphan GPCR GPR161 demonstrates that ubiquitination of ciliary GPCRs is required for their regulated exit from cilia. Furthermore, targeting a UbK63-specific deubiquitinase to cilia blocks the exit of GPR161, SSTR3, and Smoothened (SMO) from cilia. Finally, ubiquitinated proteins accumulate in cilia of mammalian photoreceptors and Chlamydomonas cells when BBSome function is compromised. We conclude that Ub chains mark GPCRs and other unwanted ciliary proteins for recognition by the ciliary exit machinery.

Conservation regulations are instrumental for effective nature preservation, but several compliance and implementation failures jeopardize the achievement of their objectives, with strong potential to erode their legitimacy. Understanding how such deficits impact on stakeholders' perceptions is a matter of concern in pursuing truly effective tools. Here, using as case study the heterogeneous implementation of EU sanitary regulations which allow livestock carcasses to be left in situ in the Iberian Peninsula, we evaluated how uneven implementation affects farmers' perceptions towards scavengers. We interviewed 109 farmers at the border between Spain, with designated Scavenger Feeding Zones (SFZs), and Portugal, where SFZs are still under way. We detected a deficit in the implementation of European sanitary regulation and a low knowledge of this legislation by farmers (10%), which led to marked differences between countries in the perception of farmers on scavengers. Despite being expected to benefit from SFZs, Spanish farmers valued scavengers worse than Portuguese farmers. This unexpected outcome would be mediated by ca. 95% of the Spanish farmers interviewed (n = 48) still using the carcass collection system set after the outbreak of the mad cow disease, instead of adopting the new rules allowing carcasses abandonment at SFZs. Contrastingly, ca. 28% of the Portuguese farmers (n = 61) left livestock carcasses in the field without official approval. Our results support the initial hypothesis of more positive perceptions of scavengers by farmers leaving livestock carcasses in situ, while warning against assuming effective implementation of sanitary regulations in reality. Worryingly, as illustrated by the negative perception of Spanish farmers towards vultures, these implementation failures could turn out to an emergent farmer-wildlife conflict, which can jeopardize scavenger conservation., This research was partially financed by the LIFE Rupis project (LIFE14 NAT/PT/000855) funded by EU LIFE Programme. J.V.L.B. was supported by a Ramón & Cajal research contract (RYC-2015-18932) from the Spanish Ministry of Economy, Industry and Competitiveness.

Cilia are microtubule-based, cell-surface projections whose machinery is evolutionarily conserved. In vertebrates, cilia are observed on almost every cell type and are either motile or immotile. Immotile sensory, or primary cilia, are responsive to extracellular ligands and signals. Cilia can be thought of as compartments, functionally distinct from the cell that provide an environment for signalling cascades. Hedgehog is a critical developmental signalling pathway which is functionally linked to primary cilia in vertebrates. The major components of the vertebrate Hedgehog signalling pathway dynamically localize to the ciliary compartment and ciliary membrane. Critically, G-protein coupled receptor (GPCR) Smoothened, the obligate transducer of the pathway, is enriched and activated in the cilium. While smoothened is the most intensely studied ciliary receptor, many GPCRs localize within cilia. Understanding the link between Smoothened and cilia defines common features, and distinctions, of GPCR signalling within the primary cilium. The cilium is an ideal environment for G-protein coupled receptor signalling. The most intensively studied ciliary G-protein coupled receptor, Smoothened, provides a template for understanding principles governing receptor activation within the cilium and the resulting signalling cascade.

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss ofArl13bdisrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358Aprotein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showedArl13bV358A/V358Amice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia,Arl13bV358A/V358Acells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.

Background Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. Methods We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. Results Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. Conclusions These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Smoothened (Smo) is the essential transducer of Sonic hedgehog (Shh) signaling, which regulates cell fate and proliferation during embryogenesis. We identified a novel mouse mutant, cabbie (cbb), and found that its cause is a missense mutation in Smo. We showed the Smo(cbb) mutation is insensitive to the Shh agonist SAG, perhaps due to the disruption of SAG binding. We characterized Smo(cbb) for defects in craniofacial and skeletal development, as well as neural tube patterning, and revealed Smo(cbb) affected processes that require the highest levels of Shh activity. Smo is normally enriched in cilia upon Shh stimulation; however, we detected inefficient enrichment of Smo in Smo(cbb) mutants whether we stimulated with Shh or SAG. Taken together, our data suggest that the highest levels of vertebrate Hedgehog signaling activity require efficient Smo ciliary enrichment.

Adolescent rats display reduced sensitivity to many dysphoria-related effects of alcohol (ethanol) including motor ataxia and sedative hypnosis, but the underlying neurobiological factors that contribute to these differences remain unknown. The cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) pathway, particularly the type II regulatory subunit (RII), has been implicated in ethanol-induced molecular and behavioral responses in adults. Therefore, the current study examined cerebral cortical PKA in adolescent and adult ethanol responses. With the exception of early adolescence, PKA RIIα and RIIβ subunit levels largely did not differ from adult levels in either whole cell lysate or P2 synaptosomal expression. However, following acute ethanol exposure, PKA RIIβ P2 synaptosomal expression and activity were increased in adults, but not in adolescents. Behaviorally, intracerebroventricular administration of the PKA activator Sp-cAMP and inhibitor Rp-cAMP prior to ethanol administration increased adolescent sensitivity to the sedative-hypnotic effects of ethanol compared to controls. Sp-cAMP was ineffective in adults whereas Rp-cAMP suggestively reduced loss of righting reflex (LORR) with paralleled increases in blood ethanol concentrations. Overall, these data suggest that PKA activity modulates the sedative/hypnotic effects of ethanol and may potentially play a wider role in the differential ethanol responses observed between adolescents and adults.

Electrical stimulation of the lateral hypothalamus can motivate feeding or can serve as a reward in its own right. It remains unclear whether the same or independent but anatomically overlapping circuitries mediate the two effects. Electrical stimulation findings implicate medial forebrain bundle (MFB) fibers of passage in both effects, and optogenetic studies confirm a contribution from fibers originating in the lateral hypothalamic area and projecting to or through the ventral tegmental area. Here we report that optogenetic activation of ventral tegmental fibers from cells of origin in more anterior or posterior portions of the MFB failed to induce either reward or feeding. The feeding and reward induced by optogenetic activation of fibers from the lateral hypothalamic cells of origin were influenced similarly by variations in stimulation pulse width and pulse frequency, consistent with the hypothesis of a common substrate for the two effects. There were, however, several cases where feeding but not self-stimulation or self-stimulation but not feeding were induced, consistent with the hypothesis that distinct but anatomically overlapping systems mediate the two effects. Thus while optogenetic stimulation provides a more selective tool for characterizing the mechanisms of stimulation-induced feeding and reward, it does not yet resolve the question of common or independent substrates.

The rate and degree to which an individual develops tolerance to the behavioral and subjective effects of alcohol (ethanol) is thought to be a major predictive factor for the development of alcoholism. In general, organisms vulnerable to the development of behavioral tolerance (ex. ethanol-induced ataxia), drink and prefer ethanol more than those that are not (Le and Kiianmaa, 1988, Newlin and Thomson, 1990, Waller et al., 1983). For example, the ‘Alko Alcohol’ (AA) line of rats selectively bred for high ethanol consumption have been shown to develop larger degrees of tolerance to several behavioral indices compared to their low drinking ‘Alko Non-Alcohol’ (ANA) counterparts (Le and Kiianmaa, 1988). Similarly, two inbred strains of mice known to differ widely on many ethanol related behavioral phenotypes, the C57BL/6J (B6) and DBA/2J (D2), have been shown to possess marked differences in their ability to develop tolerance to many of these same behaviors (Grieve et al., 1979, Tabakoff and Ritzmann, 1979). Nevertheless, very few studies have evaluated the ability of voluntary ethanol consumption to produce tolerance to behaviors known to be directly affected by that intake. In order to evaluate the development of tolerance to voluntary ethanol consumption as mentioned above, an animal model capable of eliciting consistent quantifiable behavioral impairment is a logical starting point. Obviously, evaluating the ability of voluntary ethanol consumption to produce tolerance in animals that generally avoid ethanol all together (ex. DBA/2J mice) is not a viable goal. Fortunately, a mouse model of binge-like ethanol intake, dubbed ‘Drinking in the Dark’ (DID; (Rhodes et al., 2005), has recently been developed whereby C57BL/6J (B6) mice voluntarily consume large quantities of 20% unsweetened ethanol solution when given limited access during peak arousal. Acutely, these high ethanol intakes elicit BEC’s ≥ 80 mg/dl and lead to motor impairment as indexed by the balance beam and rotarod tests (Rhodes et al., 2007), as well as alterations in locomotor behavior (unpublished observation and current data). There are several notable benefits to the DID model that make it ideal for studying the development of tolerance. Most importantly, animals will consistently and voluntarily drink-to-intoxication for at least 20 consecutive days (Boehm et al., 2008) without water deprivation or lengthy fading procedures requiring artificial sweeteners or ramping of ethanol concentration. Additionally, because ethanol consumption occurs at a very specific time interval manipulated by the experimenter, knowing precisely when to examine varying degrees of tolerance is very straightforward. For example, because animals will consume large quantities of ethanol on their very first 2-hour access session, evaluating the development of rapid tolerance (defined as a decrease in the effect of a drug 24 hours following a single ethanol exposure) is relatively simple. Additionally, the consistency of binge-like behavior observed using DID makes this model ideal for the evaluation of chronic tolerance; defined as a decrease in the effect of a drug following repeated ethanol exposures. The primary goal of this study was to evaluate the ability of the DID model to produce rapid and/or chronic tolerance to its ataxic effects. We also took the opportunity to assay for alterations in locomotor behavior and then followed up on these results with an assessment of DID-induced locomotor effects. We hypothesized that ethanol intake using DID would induce acute ataxic and locomotor effects and that additional DID ethanol exposure would lead to the development of rapid and chronic tolerance to these effects.

Animal models of prenatal ethanol exposure are necessary to more fully understand the effects of ethanol on the developing embryo/fetus. However, most models employ procedures that may produce additional maternal stress beyond that produced by ethanol alone. We employed a daily limited-access ethanol intake model called Drinking in the Dark (DID) to assess the effects of voluntary maternal binge-like ethanol intake on the developing mouse. Evidence suggests that binge exposure may be particularly harmful to the embryo/fetus, perhaps due to the relatively higher blood ethanol concentrations achieved. Pregnant females had mean daily ethanol intakes ranging from 4.2 to 6.4 g/kg ethanol over gestation, producing blood ethanol concentrations ranging from 115 to 182 mg/dL. This level of ethanol intake produced behavioral alterations among adolescent offspring that disappeared by adulthood, including altered sensitivity to ethanol's hypnotic actions. The DID model may provide a useful tool for studying the effects of prenatal ethanol exposure in mice.

The lateral habenula (LHb) is involved in reward and aversion and is reciprocally connected with dopamine (DA)-containing brain regions, including the ventral tegmental area (VTA). We used a multidisciplinary approach to examine the properties of DA afferents to the LHb in the rat. We find that >90% of VTA tyrosine hydroxylase (TH) neurons projecting to the LHb lack vesicular monoamine transporter 2 (VMAT2) mRNA, and there is little coexpression of TH and VMAT2 protein in this mesohabenular pathway. Consistent with this, electrical stimulation of LHb did not evoke DA-like signals, assessed with fast-scan cyclic voltammetry. However, electrophysiological currents that were inhibited by L741,742, a DA-D4-receptor antagonist, were observed in LHb neurons when DA uptake or degradation was blocked. To prevent DA activation of D4receptors, we repeated this experiment in LHb slices from DA-depleted rats. However, this did not disrupt D4receptor activation initiated by the dopamine transporter inhibitor, GBR12935. As the LHb is also targeted by noradrenergic afferents, we examined whether GBR12935 activation of DA-D4receptors occurred in slices depleted of norepinephrine (NE). Unlike DA, NE depletion prevented the activation of DA-D4receptors. Moreover, direct application of NE elicited currents in LHb neurons that were blocked by L741,742, and GBR12935 was found to be a more effective blocker of NE uptake than the NE-selective transport inhibitor nisoxetine. These findings demonstrate that NE is released in the rat LHb under basal conditions and that it activates DA-D4receptors. Therefore, NE may be an important regulator of LHb function.

AMPA receptor GluA2 subunits are strongly implicated in cognition, and prior work suggests that these subunits may be regulated by atypical protein kinase C (aPKC) isoforms. The present study assessed whether hippocampal and cortical AMPA receptor GluA2 subunit regulation may be an underlying factor in known age-related differences to cognitive-impairing doses of ethanol, and if aPKC isoforms modulate such responses. Hippocampal AMPA receptor GluA2 subunit, protein kinase Mζ (PKMζ), and PKCι/λ expression were elevated during adolescence compared to adults. 1 h following a low-dose (1.0-g/kg) ethanol exposure, hippocampal AMPA receptor GluA2 subunit serine 880 phosphorylation was decreased in adolescents, but was increased in adults. Age-dependent changes in GluA2 subunit phosphorylation were paralleled by alterations in aPKC isoforms, and zeta inhibitory peptide (ZIP) administration prevented ethanol-induced increases in both in adults. Ethanol-induced changes in GluA2 subunit phosphorylation were associated with delayed regulation in synaptosomal GluA2 subunit expression 24 h later. A higher ethanol dose (3.5-g/kg) failed to elicit changes in most measures in the hippocampus at either age. Similar to the hippocampus, analysis of cerebral cortical tissue also revealed age-related declines. However, no demonstrable effects were found following a low-dose ethanol exposure at either age. High-dose ethanol exposure reduced adolescent GluA2 subunit phosphorylation and aPKC isoform expression that were again accompanied by delayed reductions in synaptosomal GluA2 subunit expression. Together, these results suggest that GluA2-containing AMPA receptor modulation by aPKC isoforms is age-, region- and dose-dependently regulated, and may potentially be involved in developmentally regulated ethanol-induced cognitive impairment and other ethanol behaviors.

The reaction of N,N -diethylpropargylamine with increasing amounts of the complex [Ni(NCS) 2 (PPh 3 ) 2 ] leads to π-conjugated organometallic polymers containing Ni(II). XPS measurements show the presence of paramagnetic metal centers, which coordinate two NCS ligands, one phosphine and NEt 2 pendant group of the polyenic chain. The number of Ni-bonded atoms increases by increasing the complex/monomer molar ratio. The organometallic polymers are promising materials for humidity sensors, giving reproducible results for various subsequent cycles of relative humidity variations in the range 60–90%.

Ethanol is commonly used and abused during adolescence. Although adolescents display differential behavioral responses to ethanol, the mechanisms by which this occurs are not known. The protein kinase C (PKC) pathway has been implicated in mediating many ethanol-related effects in adults, as well as gamma-aminobutyric acid (GABA(A)) receptor regulation.The present study was designed to characterize cortical PKC isoform and GABA(A) receptor subunit expression during adolescence relative to adults as well as assess PKC involvement in ethanol action.Novel PKC isoforms were elevated, while PKCγ was lower during mid-adolescence relative to adults. Whole-cell lysate and synaptosomal preparations correlated for all isoforms except PKCδ. In parallel, synaptosomal GABAA receptor subunit expression was also developmentally regulated, with GABA(A)R δ and α4 being lower while α1 and γ2 were higher or similar, respectively, in adolescents compared to adults. Following acute ethanol exposure, synaptosomal novel and atypical PKC isoform expression was decreased only in adolescents. Behaviorally, inhibiting PKC with calphostin C, significantly increased ethanol-induced loss of righting reflex (LORR) in adolescents but not adults, whereas activating PKC with phorbol dibutyrate was ineffective in adolescents but decreased LORR duration in adults. Further investigation revealed that inhibiting the cytosolic phospholipase A2/arachidonic acid (cPLA2/AA) pathway increased LORR duration in adolescents, but was ineffective in adults.These data indicate that PKC isoforms are variably regulated during adolescence and may contribute to adolescent ethanol-related behavior. Furthermore, age-related differences in the cPLA2/AA pathway may contribute to ethanol's age-related effects on novel and atypical PKC isoform expression and behavior.

Kappa-opioid receptor (KOR) agonists have dysphoric properties in humans and are aversive in rodents. This has been attributed to the activation of KORs within the mesolimbic dopamine (DA) system. However, the role of DA in KOR-mediated aversion and stress remains divisive as recent studies have suggested that activation of KORs on serotonergic neurons may be sufficient to mediate aversive behaviors. To address this question, we used conditional knock-out (KO) mice with KORs deleted on DA neurons (DAT(Cre/wt)/KOR(loxp/loxp), or DATCre-KOR KO). In agreement with previous findings, control mice (DAT(Cre/wt)/KOR(wt/wt) or WT) showed conditioned place aversion (CPA) to the systemically administered KOR agonist U69,593. In contrast, DATCre-KOR KO mice did not exhibit CPA with this same agonist. In addition, in vivo microdialysis showed that systemic U69,593 decreased overflow of DA in the nucleus accumbens (NAc) in WT mice, but had no effect in DATCre-KOR KO mice. Intra- ventral tegmental area (VTA) delivery of KORs using an adeno-associated viral gene construct, resulted in phenotypic rescue of the KOR-mediated NAc DA response and aversive behavior in DATCre-KOR KO animals. These results provide evidence that KORs on VTA DA neurons are necessary to mediate KOR-mediated aversive behavior. Therefore, our data, along with recent findings, suggest that the neuronal mechanisms of KOR-mediated aversive behavior may include both dopaminergic and serotonergic components.

Riassunto La flora marolichenica dell'Isola di Zannone e stata studiata secondo un approccio floristico ed ecologico. Essa e costituita da 29 specie, 21 delle quali trovate per la prima volta a Zannone e 15 mai segnalate prima in alcuna isola dell'Arcipelago Pontino. I reperti piu interessanti sono costituiti da 7 specie del genere Ramalina, quasi tutte rare in Italia, e da 6 entita in via di estinzione, minacciate o vulnerabili (NIMIS, 1992). Rispetto a quanto precedentemente noto, risultano non ritrovati 12 taxa: 5 sono da ritenersi frutto di errate segnalazioni, mentre altri 4, particolarmente igrofitici, possono ritenersi estinti o fortemente diradati nell'Isola a causa di un graduale inaridimento climatico. Questo fenomeno e confermato anche dalla variazione della media degli indici di igrofitismo delle specie precedentemente note rispetto a quella della attuale flora macrolichenica.

Protein kinases are implicated in neuronal cell functions such as modulation of ion channel function, trafficking and synaptic excitability. Both protein kinase C (PKC) and A (PKA) are involved in regulation of γ-aminobutyric acid type A (GABA-A) receptors through phosphorylation. However, the role of PKA in regulating GABA-A receptors following acute ethanol exposure is not known. The present study investigated the role of PKA in ethanol effects on GABA-A receptor α1 subunit expression in the P2 synaptosomal fraction of the rat cerebral cortex. Additionally, GABA-related behaviors were also examined. Rats were administered ethanol (2.0 – 3.5 g/kg) or saline and PKC, PKA and GABA-A receptor α1 subunit levels were measured by Western blot analysis. Ethanol (3.5 g/kg) transiently increased GABA-A receptor α1 subunit expression and PKA RIIβ subunit expression at similar time points whereas PKA RIIα was increased at later time points. In contrast, PKC isoform expression remained unchanged. Notably, the moderate ethanol dose (2.0g/kg) had no effect on GABA-A α1 subunit levels although PKA RIIα and RIIβ were increased at 10 and 60 minutes, when PKC isozymes are also known to be elevated. To determine if PKA activation was responsible for the ethanol-induced elevation of GABA-A α1 subunits, the PKA antagonist H89 was administered to rats prior to ethanol exposure. H89 administration prevented ethanol-induced increases in GABA-A receptor α1 subunit expression. Moreover, increasing PKA activity intracerebroventricularly with Sp-cAMP prior to a hypnotic dose of ethanol increased ethanol-induced loss of righting reflex duration. This effect appears to be mediated in part by GABA-A receptors as increasing PKA activity also increased the duration of muscimol-induced loss of righting reflex. Overall these data suggest that PKA mediates ethanol-induced GABA-A receptor expression and contributes to ethanol behavioral effects involving GABA-A receptors.

Tolerance to the behavioral and subjective effects of alcohol (ethanol) is thought to be a major predictive factor for the development of alcoholism. Evidence from rodent models has supported this view with those animals most likely to develop tolerance generally drinking and preferring ethanol more so than those resistant to it. Despite this evidence, very little is known about the behavioral relationships between ethanol-induced tolerance and consumption. The goal of this study was to evaluate the development of tolerance to the ataxic effects of ethanol using a mouse model of binge-like intake dubbed "Drinking in the Dark" (DID; Physiol Behav 2005, 84:53-63). We hypothesized that mice would become tolerant to the ataxic effects of ethanol as this behavior is known to be altered at the blood ethanol concentrations reached using this model (≥80 mg/dl).To evaluate this, we gave daily DID ethanol or water access sessions to male C57BL/6J (B6) mice and monitored ataxia (and in some cases locomotion) at various time points.In general, mice given 14 consecutive days of ethanol access displayed tolerance to the ataxic effects of ethanol compared to water-drinking controls. These effects were coupled with alterations in locomotor behavior and in some cases differences in ethanol pharmacokinetics.Thus, we can conclude that tolerance to the behavioral effects of binge-like ethanol intake might play a key role in the daily maintenance of this behavior and that these effects may be evidence of important neuroadaptations involved in the development of alcoholism.

Nicotine is defined as substance which provokes addiction because it creates both physiological and biochemical modifications in the nervous system stimulating the activity of dopaminergic neurons releasing dopamine in the areas of the brain that control pleasure. In this paper, after a short overview of neurobiological and cellular mechanisms involved in the pathway of nicotine addiction, the main therapies, used in order to provide support to smokers who decide to reduce their cigarette consumption or to quit smoking, are examined. These therapies can be enclosed in the following categories: nicotine replacement therapy (NRT), non-nicotine pharmacological therapy (NNPT), psychological-behavioural therapies (PBT), alternative therapies (AT). In this work the advantages and disadvantages of various therapies are analysed, assessing the criteria found in literature. Results from randomised and controlled clinical studies which examine some of these therapies, alone or in association, also related to relapse time are reported. In conclusion, results of this analysis confirm that, as well as therapies and their treatment time, psychological support and personal motivation are indispensable for successful smoking cessation.

Objective To produce recommendations for children aged 2–5 years, considering the specificities of this age group in relation to nutritional needs and capacity of food intake. Methods Individual energy requirements were estimated using the formulae for calculating the Recommended Dietary Intake. Taken as reference data on weight and height, by age and sex, referring to the 50th percentile established by the multicenter study of growth references developed by the WHO (2006). After obtaining the total energy, we calculated its mean and SD for each year studied. The minimum and maximum portions related to the lower and higher energy values established were adapted in the Food Guide Pyramid developed by Philippi et al , 1996. Results The average total energy was around 1000, 1300 and 1400 kcal for two, three and 4 years old children, respectively. Minimum and maximum portions were: 4 to 7 servings for the group of cereals, breads, tubers and roots, 3 to 4 for vegetables, 4 to 5 for fruit and 3 servings for milk and milk products, 2 for meat and eggs; 1 part for the group of pulses, 1 to 2 for oils and fats, and 1 to 2 servings for sugars and sweets. Conclusion The results of this study can be applied in nutritionist9s professional practice and to planning public policies aimed at ensuring the adequate provision of nutritional support to children in the age group studied.

SUMMARYThree families from Calabria, whose members were related to each other have been studied. In the first there were both the thalas-semic and the drepanocytic traits separately in its members; in the second the thalassemic trait and in the third the drepanocytic trait. From the marriage of individuals with thalassemic trait from the first and the second family a child was born with Cooley's disease, from the marriage of drepanocytic individuals from the first and the third family a drepanocytic girl was born.The clinical, hematologic and genetic characteristics of the 19 members of these families are reported and the distribution of the pathologic features is discussed.