Littérature scientifique sur le sujet « Orexin 1 receptor »

Orexins are peptide neurotransmitters which are produced in the lateral and posterior part of the hypothalamus in the brain. There are two Orexin receptors which has been identified till date viz. Orexin 1 (OX 1) and Orexin 2 (OX 2 receptor).

Abstract Hypothalamic prepro-orexin as well as pituitary and adrenal orexin receptors are gender-specifically expressed. To assess the regulation by gonadal steroids, we investigated the effect of 17β-estradiol in female and of testosterone in male rats on prepro-orexin and orexin receptor mRNA expression. Rats were either sham-operated or gonadectomized and subsequently treated with placebo, 17β-estradiol, or testosterone for 21 d. Tissue mRNA levels of prepro-orexin, orexin type-1 (OX1), and orexin type-2 (OX2) receptors were measured using quantitative real-time RT-PCR. In female rats, pituitary OX1 receptor mRNA levels were increased 12-fold after ovariectomy compared with sham- operated rats. The increase of pituitary OX1 receptor mRNA was inhibited by treatment with 17β-estradiol. Adrenal mRNA levels of OX2 receptors in ovariectomized rats were increased 2-fold compared with sham-operated rats and were also reduced by treatment with 17β-estradiol. In male rats, orchidectomy increased the mRNA levels of pituitary OX1 receptors compared with sham-operated rats. In contrast, adrenal OX2 receptor mRNA was reduced after orchidectomy. Testosterone treatment reversed the effect of orchidectomy on pituitary OX1 and adrenal OX2 receptors. In the hypothalamus, no differences were found in the mRNA levels of prepro-orexin, OX1, and OX2 receptors between sham-operated, placebo-treated, and steroid-treated female or male rats. Our results indicate that gonadal steroids differentially regulate pituitary OX1 receptors and adrenal OX2 receptors in male and female rats and may contribute to specific sex- dependent neuroendocrine and endocrine actions of orexins.

Orexins/hypocretins exert cardiovascular effects which are centrally mediated. In the present study, we tested whether orexins and their receptors may also act in an autocrine/paracrine manner in the heart exerting direct effects. Quantitative reverse transcription-PCR (RT-PCR), immunohistochemical and Western blot analyses revealed that the rat heart expresses orexins and orexin receptors (OXR). In isolated rat cardiomyocytes, only orexin-B (OR-B) caused an increase in contractile shortening, independent of diastolic or systolic calcium levels. A specific orexin receptor-2 (OX2R) agonist ([Ala11, d-Leu15]-Orexin B) exerted similar effects as OR-B, whereas a specific orexin receptor-1 (OX1R) antagonist (SB-408124) did not alter the responsiveness of OR-B. Treatment of the same model with OR-B resulted in a dose-dependent increase in myosin light chain and troponin-I (TnI) phosphorylation. Following ischaemia/reperfusion in the isolated Langendorff perfused rat heart model, OR-B, but not OR-A, exerts a cardioprotective effect; mirrored in an in vivo model as well. Unlike OR-A, OR-B was also able to induce extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2) and Akt phosphorylation in rat myocardial tissue and ERK1/2 phosphorylation in human heart samples. These findings were further corroborated in an in vivo rat model. In human subjects with heart failure, there is a significant negative correlation between the expression of OX2R and the severity of the disease clinical symptoms, as assessed by the New York Heart Association (NYHA) functional classification. Collectively, we provide evidence of a distinct orexin system in the heart that exerts a cardioprotective role via an OR-B/OX2R pathway.

Abstract Two recently discovered hypothalamic peptides, orexin-A and orexin-B, play a role as mediators in the central mechanisms that regulate feeding behavior and sleep control. These peptides bind and activate two orexin receptors that belong to the G-protein coupled receptor superfamily. Morphological studies have detected mRNA expression of orexin receptors exclusively in the rat central nervous system. In this paper we demonstrate a strong level of expression of orexin receptor 1 and 2 in the adrenal medulla of the rat by RT-PCR immunohistochemistry. The results of the present study provide the first evidence showing that the adrenal medulla expresses orexin receptors, and thus appears to be a target tissue for orexins. This could open a new loop in which the central and autonomous nervous system may be involved in body weight homeostasis and sleep control.

Orexin-A and orexin-B, via their receptors orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R) have been shown to play a role in the regulation of feeding, body weight, and energy expenditure. Adipose tissue also contributes significantly to the maintenance of body weight by interacting with a complex array of bioactive peptides; however, there are no data as yet on the expression of orexin components in adipose tissue. We, therefore, analyzed the expression of OX1R and OX2R in human adipose tissue and determined functional responses to orexin-A and orexin-B. OX1R and OX2R mRNA expression was detected in subcutaneous (s.c.) and omental adipose tissue and in isolated adipocytes. Protein for OX1R and OX2R was also detected in whole adipose tissue sections and lysates. Treatment with orexin-A, and orexin-B (100 nM, 24 h) resulted in a significant increase in peroxisome proliferator-activated receptors γ-2 mRNA expression in s.c. adipose tissue (P < 0.05). Hormone sensitive lipase mRNA was significantly reduced in omental adipose tissue with orexin-A and orexin-B treatment (P < 0.05). Glycerol release from omental adipose tissue was also significantly reduced with orexin-A treatment (P < 0.05). These findings demonstrate for the first time the presence of functional orexin receptors in human adipose tissue and suggest a role for orexins in adipose tissue metabolism and adipogenesis.

Orexin peptides comprise two neuropeptides, orexin A and orexin B, that bind two G-protein coupled receptors (GPCRs), orexin receptor 1 (OXR1) and orexin receptor 2 (OXR2). Although cell bodies that produce orexin peptides are localized in a small area comprising the lateral hypothalamus and adjacent regions, orexin-containing fibres project throughout the neuraxis. Although orexins were initially described as peptides that regulate feeding behaviour, research has shown that orexins are involved in diverse functions that range from the modulation of autonomic functions to higher cognitive functions, including reward-seeking, behaviour, attention, cognition, and mood. Furthermore, disruption in orexin signalling has been shown in mood disorders that are associated with low hedonic tone or anhedonia, including depression, anxiety, attention deficit hyperactivity disorder, and addiction. Notably, projections of orexin neurons overlap circuits involved in the modulation of hedonic tone. Evidence shows that orexins may potentiate hedonic behaviours by increasing the feeling of pleasure or reward to various signalling, whereas dysregulation of orexin signalling may underlie low hedonic tone or anhedonia. Further, orexin appears to play a key role in regulating behaviours in motivationally charged situations, such as food-seeking during hunger, or drug-seeking during withdrawal. Therefore, it would be expected that dysregulation of orexin expression or signalling is associated with changes in hedonic tone. Further studies investigating this association are warranted.

Abstract Orexins are hypothalamic neuropeptides primarily involved in the regulation of food intake and arousal states. In addition, a role for orexins as central neuroendocrine modulators of reproductive function has recently emerged. Prepro-orexin and orexin type-1 receptor mRNAs have been detected in the rat testis. This raises the possibility of additional peripheral actions of orexins in the control of reproductive axis, which remains so far unexplored. To analyze the biological effects and mechanisms of action of orexins in the male gonad, we evaluated testicular expression of orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) mRNAs in different experimental settings and the effect of orexin-A on testicular testosterone (T) secretion. Persistent expression of OX1R mRNA was demonstrated in the rat testis throughout postnatal development. In contrast, OX2R transcript was not detected at any developmental stage. Expression of OX1R mRNA persisted after selective elimination of mature Leydig cells and was detected in isolated seminiferous tubules at defined stages of the seminiferous epithelial cycle. In addition, testicular OX1R mRNA expression appeared to be under hormonal regulation; it was reduced by long-term hypophysectomy and partially restored by FSH replacement, whereas down-regulation was observed after exposure to increasing doses of the ligand in vitro. Moreover, OX1R mRNA expression was sensitive to neonatal imprinting by estrogen. Finally, orexin-A, in a dosedependent manner, significantly increased basal, but not human choriogonadotropin-stimulated, T secretion in vitro. A similar stimulatory effect was observed in vivo after intratesticular administration of orexin-A. In conclusion, our present results provide the first evidence for the regulated expression of OX1R mRNA and functional role of orexin-A in the rat testis. Overall, our data are suggestive of a novel site of action of orexins in the control of male reproductive axis.

Orexins, also called hypocretins, are newly discovered hypothalamic peptides that are thought to be involved in various physiological functions. In spite of the fact that orexin receptors, especially orexin receptor 2, are abundant in the hypothalamic paraventricular nucleus (PVN), the effects of orexins on PVN neurons remain unknown. Using a whole cell patch-clamp recording technique, we investigated the effects of orexin-B on PVN neurons of rat brain slices. Bath application of orexin-B (0.01–1.0 μM) depolarized 80.8% of type 1 ( n = 26) and 79.2% of type 2 neurons tested ( n = 24) in the PVN in a concentration-dependent manner. The effects of orexin-B persisted in the presence of TTX (1 μM), indicating that these depolarizing effects were generated postsynaptically. Addition of Cd2+(1 mM) to artificial cerebrospinal fluid containing TTX (1 μM) significantly reduced the depolarizing effect in type 2 neurons. These results suggest that orexin-B has excitatory effects on the PVN neurons mediated via a depolarization of the membrane potential.

Abstract In humans and rat, orexins orchestrate divergent actions through their G protein-coupled receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Orexins also play an important physiological role in mouse, but the receptors through which they function are not characterized. To characterize the physiological role(s) of orexins in the mouse, we cloned and characterized the mouse orexin receptor(s), mOX1R and mOX2R, using rapid amplification of cDNA (mouse brain) ends, RT-PCR, and gene structure analysis. The mOX1R cDNA encodes a 416-amino acid (aa) receptor. We have identified two alternative C terminus splice variants of the mOX2R; mOX2αR (443 aa) and mOX2βR (460 aa). Binding studies in human embryonic kidney 293 cells transfected with mOX1R, mOX2αR, and the mOX2βR revealed specific, saturable sites for both orexin-A and -B. Activation of these receptors by orexins induced inositol triphosphate (IP3) turnover. However, human embryonic kidney 293 cells transfected with mOXRs demonstrated no cAMP response to either orexin-A or orexin-B challenge, although forskolin and GTPγS revealed a dose-dependent increase in cAMP. Although, orexin-A and -B showed no difference in binding characteristics between the splice variants; interestingly, orexin-B led to an increase in IP3 production at all concentrations in the mOX2βR variant. Orexin-A, however, showed no difference in IP3 production between the two variants. Additionally, in the mouse, we demonstrate that these splice variants are distributed in a tissue-specific manner, where OX2αR mRNA was undetectable in skeletal muscle and kidney. Moreover, food deprivation led to a greater increase in hypothalamic mOX2βR gene expression, compared with both mOX1R and mOX2αR. This potentially implicates a fundamental physiological role for these splice variants.

Abstract Orexin receptors play a role in regulation of sleep-wake-rhythm, food intake and energy homeostasis and they were long thought to be exclusively expressed in the nervous system. During the last years orexin receptors are being identified in a growing number of peripheral tissues. We have earlier detected orexin receptor 1 and 2 expression on human CD34+ blood stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seem to be restricted to the central nerve system to this date. The main downstream signaling pathways of the orexin receptors include Ca2+-dependent signaling associated with activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways. In an attempt to investigate if the receptors are functionally active in CD34+ stem and progenitor cells, we used live cell calcium imaging and stimulated purified CD34+ stem and progenitor cells with orexin A and B. Upon stimulation a massive intracellular calcium release was seen which could not been detected using cells preincubated with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the selective OX1R-Antagonist SB334867 and CD34 negative cells. Additionally, upon stimulation with orexin A and B we found ERK (1/2) activation at a maximum 3 hours following incubation with orexin A whereas no effect was seen after stimulation with orexin B. To investigate a potential impact on the functional properties of human CD34+ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. So far, no effects of orexin stimulation on the proliferation and apoptosis of CD34+ cells were apparent. Remarkably, stimulation with orexin A and B led to a significantly higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors BFU-E (burst forming unit erythrocyte) and CFU-E (colony forming unit erythrocyte). A more immature phenotype of orexin-stimulated CD34+ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significantly higher frequency of LTC-IC (long-term-culture initiating cells) indicating also a more immature phenotype of orexin-stimulated cells and a greater repopulating capacity. The selective orexin receptor antagonist SB-334867 abrogated these effects. No differences could be observed regarding the migration towards SDF-1 with and without stimulation with orexin A and B. Still, orexin A and B led to a decrease in the adhesive capacity of CD34+ stem and progenitor cells to fibronectin coated dishes. Since orexin receptors are coupled to inhibitory G-proteins (Gi/q) and stimulatory G-proteins (Gs) dependent on the tissue, we incubated CD34+ cells with the selective inhibitor of Gi – proteins pertussis toxin concurrently to stimulation with orexins and observed no differences in the adhesive capacity of CD34+ cells compared to the unstimulated controls suggesting coupling of the orexin receptor 1 and 2 to Gi – proteins rather than Gs-proteins in CD34+ cells. Given this functional impact of the orexin system on CD34+ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34+ cells or if they are humorally transported to the bone marrow cavity. Using ELISA we did not find autocrine production of orexin by CD34+ cells whereas orexin could be detected in the serum obtained by bone marrow biopsies and peripheral blood pointing rather towards a humoral delivery of orexins to CD34+ cells. Taken together, our findings indicate a functional role of the orexin system in CD34+ stem and progenitor cells.

Diversi studi riportano un coinvolgimento dei cannabinoidi e dell’orexina-A (OX-A) nella regolazione dell’appetito. I neuroni ipotalamici pro-opiomelanocortinici (POMC) inducono sazietà mediante il rilascio del peptide stimolante i melanociti di tipo α (α-MSH). I neuroni POMC esprimono sia il recettore dell’OX-A (OX-1R) che il recettore di tipo 1 dei cannabinoidi (CB1R). Il mio progetto di ricerca si basa nel verificare se la stimolazione dell’appetito da parte dell’OX-A, sia dovuta all’inibizione funzionale endocannabinoide-mediata dei neuroni POMC.Noi abbiamo dimostrato che l’assenza della leptina in topi obesi, aumenta l’espressione dell’OX-A la quale promuove il senso di fame riducendo l’espressione di POMC ed il rilascio di α-MSH. Questi processi coinvolgono la biosintesi OX-A-mediata del principale endocannabinoide espresso nel cervello, il 2-arachidonoil-glicerolo (2-AG) il quale, tramite attivazione del CB1R, promuove la fosforilazione di ERK1/2 simultaneamente all’inibizione dell’espressione del gene Pomc tramite STAT3. L’interazione CB1R/OX-1R è alla base dell’iperfagia e dell’aumento di peso dovuto all’incremento della sintesi del 2-AG mediato dall’OX-A.Infatti, la somministrazione dell’antagonista selettivo dell’OX-1R, SB334867, ostacola l’effetto oressizzante dell’OX-A. Dal punto di vista clinico, una significativa correlazione inversa è stata riscontrata tra i livelli sierici di OX-A e α-MSH in pazienti obesi di sesso maschile associata ad alterazione delle concentrazioni sieriche delle transaminasi, i principali markers di steatosi epatica. Questi risultati dimostrano un ruolo, fin adesso sconosciuto, del peptide OX-A nel promuovere il senso di fame, attraverso la diminuzione della sintesi ipotalamica del peptide POMC e del rilascio del suo prodotto α-MSH ed i cui effetti anoressizzanti sono ampiamente documentati dalla letteratura scientifica.
Several studies reveal the involvement of cannabinoids and orexin-A (OX-A) in the regulation of food intake. Starting from this basis, we hypothesized that OX-A regulates the pro-opiomelanocortin (POMC) neurons, which are the main cannabinoid target in the hypothalamus. POMC neurons induce satiety through the synthesis and release of α-MSH (melanocyte-stimulating hormone). Since both OX-A receptor-1 (OX-1R) and cannabinoid type 1 receptor (CB1R) are expressed in POMC neurons we sought to investigate the hypothesis that OX-A promotes appetite by inhibition of Pomc gene transcription (through the CB1R signaling) and, in exaggerated manner, during obesity. At this purpose we used murine obese models (ob/ob and HFD) and lean mice injected with OX-A. In obese mice, we found that leptin signal deficiency increases OX-A expression in fibers projecting to arcuate nucleus and promotes appetite by lowering POMC expression and α-MSH release. These effects occur by OX-A-mediated biosynthesis of the endocannabinoid 2-arachidonoyl glycerol (2-AG), the activation of ERK1/2 which inhibits its target STAT3 finally resulting in the reduction of the Pomc gene transcription. In lean mice, the injection of OX-A induces hyperphagia and weight gain, which are both prevented by the selective OX-1R antagonist SB334867. Notably, in human obese subjects, we found an inverse correlation between OX-A and α-MSH serum levels associated with alterations of transaminases. These results reveal an unknown role of OX-A to promote appetite acting through a severe reduction in POMC expression and POMC-derived α-MSH release, which are the main inhibitors of appetite.

L'obesità è un problema mondiale, che colpisce la salute delle persone e gravare sistemi sanitari. Dal punto di vista del biologo è una complessa interazione di endocrine e neurali meccanismi sottostanti assunzione di cibo, in particolare, una delle sue forme patologiche porta all'obesità. È interessante notare che i pazienti così come modelli animali affetti da anomalie di visualizzazione comportamento alimentare nocicezione alterato. Un modello di topo fama per l'obesità è la leptina-carente ob/ob topo, il nostro laboratorio ha dimostrato che l'innervazione prevalentemente eccitatoria dei neuroni esprimono orexin (OX-N) nell'ipotalamo laterale (LH) di topi WT è riorganizzato in favore di gli ingressi inibitori di LH di topi ob/ob. Inoltre, il rilascio delle vescicole dagli ingressi inibitori è soppressa via recettore dei cannabinoidi di tipo 1 (CB1) attivazione. Su depolarizzazione un neurone oressinergico sintetizza e "rilascia" endocannabinoidi (eCBs), molto probabilmente 2-AG, che viaggia retrograda al terminale presinaptico e attiva i recettori CB1 presinaptici, sopprimendo così il rilascio delle vescicole da questi terminali (un meccanismo chiamato: depolarizzazione-indotta soppressione di inibizione o DSI). Qui, ho dimostrato che l'innervazione eccitatoria funzionale di OX-N non differiva tra ob/ob e topi WT. Inoltre, l'attivazione dei recettori CB1 presinaptici soppresso il rilascio delle vescicole da ingressi eccitatori, sia wt e topi ob/ob, nella stessa misura. Lo squilibrio di eccitatori funzionale e ingressi inibitori in ob/ob recettori CB1 presinaptici topi, putativamente, lascia OX-N con una membrana iperpolarizzato potenziale e un'attività di cottura ridotta, su depolarizzazione, tuttavia, eCBs sarebbe sintetizzato e rilasciato, viaggi in e attivare , che si trova principalmente su fattori inibitori, sopprimendo il rilascio delle vescicole e disinibendo quindi OX-N. Neuroni oressinergico possiedono vaste proiezioni in tutto il cervello, i.a. per il sistema della dopamina mesoaccumbal e (HPA) ipotalamo-ipofisi-surrene. L'attivazione di queste due circuiti, putativamente, provoca l'aumentata assunzione di cibo visto in ob/ob mouse. Un'altra area di destinazione di OX-N è il grigio periacqueduttale (PAG), che è noto a svolgere un ruolo chiave nella nocicezione attraverso le vie discendenti antinocicettivi. E 'stato riportato che i pazienti così come modelli animali affetti da comportamento alimentare anormale mostrano anche nocicezione alterata. Inoltre, la somministrazione di orexina A (OX-A) è stata dimostrata per sopprimere le correnti postsinaptiche inibitorie in recettori CB1 modo attivazione mediata, eventualmente, causando una depolarizzazione del potenziale di membrana dei neuroni PAG e, infine, un aumento di cottura attività. Curiosamente, queste osservazioni in vitro si traducono in un comportamento, OX-Un'amministrazione nel PAG elevato la soglia del dolore nei ratti durante il test della coda-flick. ob/ob topo mostravano un elevato livello di OX-A in PAG, simile alla situazione dopo OX-A amministrazione, quindi, abbiamo ipotizzato che i neuroni PAG proiettando al midollo rostroventral (prossimo passo gerarchica dopo PAG nelle vie discendenti antinocicettivi) erano più depolarizzato e hanno un'attività di cottura superiore in topi ob/ob rispetto al peso. Infatti, bloccando il recettore orexina 1 iperpolarizzato il potenziale di membrana e riduce l'attività dei neuroni ob/ob PAG, ma non i neuroni wt PAG. Inoltre, PAG neuroni di topi ob/ob visualizzati potenziale potenziale soglia azione iperpolarizzato rispetto ai topi WT, il che significa che i neuroni ob/ob PAG sono più propensi ad avviare un potenziale d'azione di neuroni WT PAG. Così, suggerendo che l'attivazione dei neuroni PAG dal attivati ingressi oressinergico risultati in una soglia del dolore elevata ulteriormente attivando le vie discendenti antinocicettivi. In conclusione, l'interruttore di innervazione su OX-N a favore di ingressi inibitori, causato dall'assenza di leptina nel ob/ob topo, attiva OX-N inibendo loro ingressi principalmente inibitori dall'attivazione del recettore CB1 BCE-mediata, conseguente la soppressione del rilascio di vescicole (DSI). Questi disinibito OX-N attivare aree di destinazione in tutto il cervello, come il sistema della dopamina mesoaccumbal e asse HPA, modulando in tal modo il comportamento alimentare. Inoltre, le proiezioni oressinergico a PAG depolarizza il potenziale di membrana e aumenta l'attività dei neuroni PAG proiettano al midollo rostroventral, aumentando così la soglia del dolore.
Obesity is a worldwide problem, affecting peoples‘ health and burdening healthcare systems. It is a complex interaction of endocrine and neural mechanisms underlying food intake, in particular, one of its pathological forms leading to obesity. Interestingly, patients as well as animal models suffering from abnormal feeding behavior display altered nociception. A renown mouse model for obesity is the leptin-deficient ob/ob mouse, our laboratory has demonstrated that the mainly excitatory innervation of orexin-expressing neurons (OX-N) in the lateral hypothalamus (LH) of wt mice is rearranged in favor of the inhibitory inputs in LH of ob/ob mice. Furthermore, the vesicle release from the inhibitory inputs is suppressed by endocannabinoids (eCBs) activating cannabinoid receptor type 1 (CB1) activation. The eCBs, most likely 2-AG, originate from the postsynaptic terminals, where they are being synthesized and “released“ as a reaction to depolarization (a mechanism called: depolarization-induced suppression of inhibition or DSI). Here, I demonstrated that the functional excitatory innervation of OX-N did not differ between ob/ob and wt mice. Furthermore, the activation of presynaptic CB1 receptors suppressed the vesicle release from excitatory inputs, in both wt and ob/ob mice, to the same extent. The imbalance of functional excitatory and inhibitory inputs in ob/ob mice, putatively, leaves OX-N with a hyperpolarized membrane potential and a reduced firing activity. Activation of CB1 receptors, mainly located on inhibitory inputs, eventually activate OX-N by disinhibiting them. Orexinergic neurons possess vast projections throughout the brain, i.a. to the mesoaccumbal dopamine system and the hypothalamus-pituitary-adrenal (HPA) axis. The activation of these two circuits, putatively, results in the increased food intake seen in the ob/ob mouse. Another target area of OX-N is the periaqueductal gray (PAG), playing a key role in nociception via the descending antinociceptive pathways. It has been reported that patients as well as animal models suffering from abnormal feeding behavior also display altered nociception. Furthermore, the administration of orexin A (OX-A) has been demonstrated to suppress inhibitory postsynaptic currents in CB1 receptor activation-mediated way, eventually, resulting in a depolarization of the membrane potential of PAG neurons and, finally, in an increase of firing activity. Intriguingly, these in vitro observations translate to behavior, OX-A administration into PAG elevated the pain threshold in rats during the tail-flick test. ob/ob mice displayed an elevated level of OX-A in PAG, similar to the situation after OX-A administration, hence, we hypothesized that the PAG neurons projecting to the rostroventral medulla were more depolarized and have a higher firing activity in ob/ob mice compared to wt. Indeed, blocking the orexin 1 receptor hyperpolarized the membrane potential and reduced the firing activity of ob/ob PAG neurons, but not wt PAG neurons. Furthermore, ob/ob PAG neurons were more likely to initiate an action potential than wt PAG neurons. Thus, suggesting that the activation of PAG neurons by activated orexinergic inputs results in an elevated pain threshold by further activating the descending antinociceptive pathways. In conclusion, the switch of innervation onto OX-N in favor of inhibitory inputs, caused by the absence of leptin in the ob/ob mouse, activates OX-N by inhibiting their mainly inhibitory inputs by eCB-mediated CB1 receptor activation, resulting in the suppression of vesicle release (DSI). These disinhibited OX-N activate target areas throughout the brain, such as the mesoaccumbal dopamine system and HPA axis, thereby modulating feeding behavior. Furthermore, orexinergic projections to PAG depolarizes the membrane potential and increases the firing activity of PAG neurons projecting to the rostroventral medulla, thereby raising the pain threshold.

Many of the same behavioural and brain disturbances observed in addiction are also seen in obesity and binge-eating disorder. This suggests that there are shared neural substrates between substance addiction and compulsive food consumption. Food intake and appetite are regulated by numerous appetite hormones that exert their effects through brain systems involved in reward sensitivity, stress, impulsivity, and compulsivity. There is now emerging evidence that appetite hormones (e.g. ghrelin, glucagon-like peptide-1, orexin) can modulate addictive behaviours (e.g. craving) and the intake of alcohol and drugs. Therefore, there is an emerging shift into a new field of testing drugs that affect appetite hormones and their receptors in the brain, and their use in regulating the brain mechanisms that lead to relapse in addiction disorders.

Integrated pest management in modern crop protection may combine chemical and biological insecticides, particularly due to the risks to the environment and livestock arising from the massive use of non-selective chemicals. Thus, there is a need for safer alternatives, which target insects more specifically. Scorpions produce anti-insect selective polypeptide toxins that are biodegradable and non-toxic to warm-blooded animals. Therefore, integration of these substances into insect pest control strategies is of major importance. Moreover, clarification of the molecular basis of this selectivity may provide valuable information pertinent to their receptor sites and to the future design of peptidomimetic anti-insect specific substances. These toxins may also be important for reducing the current overuse of chemical insecticides if they produce a synergistic effect with conventional pesticides. Based on these considerations, our major objectives were: 1) To elucidate the three-dimensional structure and toxic-site of scorpion excitatory, "depressant, and anti-insect alpha toxins. 2) To obtain an initial view to the sodium channel recognition sites of the above toxins by generating peptide decoys through a phage display system. 3) To investigate the synergism between toxins and chemical insecticides. Our approach was to develop a suitable expression system for toxin production in a recombinant form and for elucidation of toxin bioactive sites via mutagenesis. In parallel, the mode of action and synergistic effects of scorpion insecticidal toxins with pyrethroids were studied at the sodium channel level using electrophysiological methods. Objective 1 was achieved for the alpha toxin, LqhaIT Zilberberg et al., 1996, 1997; Tugarinov et al., 1997; Froy et al., 2002), and the excitatory toxin, Bj-xtrIT (Oren et al., 1998; Froy et al., 1999; unpublished data). The bioactive surface of the depressant toxin, LqhIT2, has been clarified and a crystal of the toxin is now being analyzed (unpublished). Objective 2 was not successful thus far as no phages that recognize the toxins were obtained. We therefore initiated recently an alternative approach, which is introduction of mutations into recombinant channels and creation of channel chimeras. Objective 3 was undertaken at Riverside and the results demonstrated synergism between LqhaIT or AaIT and pyrethroids (Lee et al., 2002). Furthermore, negative cross-resistance between pyrethroids and scorpion toxins (LqhaIT and AaIT) was demonstrated at the molecular level. Although our study did not yield a product, it paves the way for future design of selective pesticides by capitalizing on the natural competence of scorpion toxins to distinguish between sodium channels of insects and vertebrates. We also show that future application of anti-insect toxins may enable to decrease the amounts of chemical pesticides due to their synergism.