Varenicline, an alpha-4 beta-2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking

Abstract Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the alpha4beta2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the alpha4beta2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Molecule Modeling of Human Pentameric alpha(7) Neuronal Nicotinic Acetylcholine Receptor and Its Interaction with its Agonist and Competitive Antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved i interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([-'H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations. one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the highaffinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmolimg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60-70 fmolimg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life. Key Words: Cholinergic muscarinic receptors-Quinuclidinyl benzilate-Corpus striaturn-Brainstem-Cerebellum. Ravikumar B. V. and Sastry P. S. Cholinergic muscarinic receptors in human fetal brain: Ontogeny of [3H]quinuclidinyl benzilate binding sites in corpus striatum, brainstem, and cerebellum. J. Neurochem. 45, 1948- 1950 (1985).

Cellular Mechanisms of Sleep Homeostasis : Studies on Brain Energy Metabolism and Aging

Sleep is governed by a homeostatic process in which the duration and quality of previous wake regulate the subsequent sleep. Active wakefulness is characterized with high frequency cortical oscillations and depends on stimulating influence of the arousal systems, such as the cholinergic basal forebrain (BF), while cessation of the activity in the arousal systems is required for slow wave sleep (SWS) to occur. The site-specific accumulation of adenosine (a by-product of ATP breakdown) in the BF during prolonged waking /sleep deprivation (SD) is known to induce sleep, thus coupling energy demand to sleep promotion. The adenosine release in the BF is accompanied with increases in extracellular lactate and nitric oxide (NO) levels. This thesis was aimed at further understanding the cellular processes by which the BF is involved in sleep-wake regulation and how these processes are affected by aging. The BF function was studied simultaneously at three levels of organization: 1) locally at a cellular level by measuring energy metabolites 2) globally at a cortical level (the out-put area of the BF) by measuring EEG oscillations and 3) at a behavioral level by studying changes in vigilance states. Study I showed that wake-promoting BF activation, particularly with glutamate receptor agonist N-methyl-D-aspatate (NMDA), increased extracellular adenosine and lactate levels and led to a homeostatic increase in the subsequent sleep. Blocking NMDA activation during SD reduced the high frequency (HF) EEG theta (7-9 Hz) power and attenuated the subsequent sleep. In aging, activation of the BF during SD or experimentally with NMDA (studies III, IV), did not induce lactate or adenosine release and the increases in the HF EEG theta power during SD and SWS during the subsequent sleep were attenuated as compared to the young. These findings implicate that increased or continuous BF activity is important for active wake maintenance during SD as well as for the generation of homeostatic sleep pressure, and that in aging these mechanisms are impaired. Study II found that induction of the inducible NO synthase (iNOS) during SD is accompanied with activation of the AMP-activated protein kinase (AMPK) in the BF. Because decreased cellular energy charge is the most common cause for AMPK activation, this finding implicates that the BF is selectively sensitive to the metabolic demands of SD as increases were not found in the cortex. In aging (study III), iNOS expression and extracellular levels of NO and adenosine were not significantly increased during SD in the BF. Furthermore, infusion of NO donor into the BF did not lead to sleep promotion as it did in the young. These findings indicated that the NO (and adenosine) mediated sleep induction is impaired in aging and that it could at least partly be due to the reduced sensitivity of the BF to sleep-inducing factors. Taken together, these findings show that reduced sleep promotion by the BF contributes to the attenuated homeostatic sleep response in aging.

Fluorescence And Nmr-Studies Of The Binding Of Cholinergic Fluorescent-Probes To Horse Serum-Cholinesterase

The interaction of the cholinergic fluorescent probes, 1-(5-dimethyl-aminoaphthalene-1-sulfonamido) ethane-2-trimethylammonium perchlorate, 1-(5-dimethylaminonaphthalene-1-sulfonamido) pentane-5-trimethylammonium tartarate and 1-(5-dimethylaminonaphthalene-1-sulfonamido) decane-10- trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.

Nikotiinireseptoreiden kautta vaikuttavat lääkeaineet nikotiiniriippuvuuden ja neurologisten sairauksien hoidossa

Neuronaaliset nikotiinireseptorit liittyvät tupakkariippuvuuden lisäksi moniin neurologisiin sairauksiin, kuten Alzheimerin tautiin, skitsofreniaan, masennukseen ja tarkkaavaisuus- ja ylivilkkaushäiriöön. Nikotiinireseptorien stimulaation on tutkimuksissa havaittu parantavan kognitiota. Useat lääkeyritykset tutkivat nikotiinireseptoriagonisteja ja -antagonisteja eri neurologisten sairauksien hoidossa. Ongelmana nikotiinireseptori-agonisteja käytettäessä on reseptorissa tapahtuva desensitisaatio. Tällöin reseptori sulkeutuu, eikä aktivoidu vaikka agonistia olisi tarjolla tai sitoutuneena reseptoriin. Varsinkin alfa7-reseptori desensitoituu hyvin nopeasti agonistialtistuksen seurauksena. Reseptorien desensitoituminen voi kliinisessä käytössä aiheuttaa lääkeaineen tehon menetyksen. Perinteisen agonistin sitoutumiskohdan lisäksi nikotiinireseptorissa sijaitsee myös muita sitoutumiskohtia, joita kutsutaan allosteerisiksi sitoutumispaikoiksi. Tutkimuksissa on havaittu, että eräät allosteerisesti sitoutuvat aineet, kuten PNU-120596, voivat vahvistaa agonistin aikaansaamaa vastetta ja/tai estää reseptorin desensitoitumista. Näitä aineita kutsutaan positiivisiksi allosteerisiksi modulaattoreiksi ja niiden ajatellaan olevan vaihtoehto desensitoitumisen aiheuttamaan tehon menetyksen ongelmaan. Nikotiinireseptorien positiivisten allosteeristen modulaattorien tarkkaa vaikutusta ja sitoutumiskohtaa reseptoriin ei vielä tarkkaan tiedetä. Tutkimuksen aiheena oli karakterisoida positiivisten allosteeristen modulaattoreiden vaikutuksia alfa7-nikotiinireseptoriin. Tutkimuksessa tarkoituksena oli käyttää hyväksi laboratoriossa aiemmin tehtyä havaintoa, jonka mukaan alfa7-nikotiinireseptorin transmembraaniosan aminohappoon tehdyn mutaation L247T seurauksena positiiviset allosteeriset modulaattorit muuttuvat agonisteiksi. Haluttiin selvittää, kuinka agonistin sitoutumiskohtaan kohdennettua mutageneesiä käyttäen tehty mutaatio W149M tai W149F vaikuttavat PNU-120596:n kykyyn toimia agonistina alfa7L247T reseptoriin. Asetyylikoliini toimi konventionaalisen agonistin mallina tutkimuksessa. Tutkimuksen toinen tavoite oli tehdä mutaatio M253Lalfa7-reseptorin transmembraaniosaan. Mutaation on todettu estävän allosteeristen potentiaattoreiden kykyä voimistaa agonistin aikaansaamaa vastetta. Tarkoitus oli tutkia millaisia vaikutuksia M253L-mutaatiolla on allosteerisen potentiaattorin kykyyn toimia agonistina L247T-mutaation sisältävään reseptoriin. Mutatoidun reseptorin mRNA mikroinjektoitiin oosyyttiin ja elektrofysiologian avulla tutkittiin ilmennettyjen reseptorien toimintaa käyttäen kahden elektrodin jännitelukitus -menetelmää. Kaikki suunnitellut mutaatiot saatiin tehtyä onnistuneesti alfa7- ja alfa7L247T-reseptoreihin. Ortosteerisen sitoutumiskohdan mutaatio villin tyypin Į7-reseptorissa vaikutti hyvin voimakkaasti joko asetyylikoliinin sitoutumiseen reseptoriin tai reseptorin toimintaan, sillä asetyylikoliinilla ei reseptorista saatu mitattua vasteita. Myöskään PNU-120596 yksinään ei saanut aikaan vasteita alfa7W149M-reseptorissa. Kaksoismutatoidussa alfa7W149M/L247T-reseptorissa puolestaan havaittiin, että asetyylikoliinin annos-vaste -kuvaaja siirtyi huomattavasti enemmän oikealle kuin PNU-120596:n, kun verrattiin annos-vaste –kuvaajia alfa7L247T ja alfa7W149M/L247T–reseptoreiden välillä. Transmembraaniosan mutaatio M253L ei vaikuttanut PNU-120596:n kykyyn toimia agonistina alfa7L247T-reseptoriin, eikä sillä ollut vaikutusta asetyylikoliinin annosvaste-kuvaajiin. Tutkimus tukee aiempia havaintoja siitä, että positiivisten allosteeristen modulaattoreiden sitoutumiskohta nikotiinireseptorissa sijaitsisi transmembraaniosassa. M253L-mutaation osalta tulokset ovat hieman ristiriidassa aiempien tulosten kanssa. L247T-mutaatio vaikuttaa hyvin voimakkaasti nikotiinireseptorin toimintaan sekä sijaitsee aminohapon M253 läheisyydessä. On mahdollista, että se peittää M253L-mutaation vaikutuksen. Toisaalta voi olla, että M253 on aminohappo, joka vaikuttaa vain reseptorivasteiden voimistumiseen eikä allosteeristen potentiaattoreiden sitoutumiseen.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Effect of constant infusion of gonadotropin releasing hormone (GnRH) agonist Buserelin and antagonist CDB 2085 A using osmotic minipumps on testicular function in adult male bonnet monkey (Macaca radiata)

The effect of chronic infusion of gonadotropic hormone agonist Buserelin or antagonist CDB 2085 A for 15 weeks via alzet minipumps in adult male bonnet monkeys was studied. Infusion of Buserelin resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours, decrease in responsiveness to injected Buserelin as judged by change in serum testosterone values from pre-injection values and decrease in sperm counts. Infusion of antagonist resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours.

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.

Cholinergic neurons within the nucleus basalis magnocellularis in bilaterally lesioned rats with 192IgG-saporin.

En el presente estudio se han estudiado las neuronas colinérgicas del núcleo basal magnocelular y sus áreas de proyección en ratas lesionadas bilateralmente con la inmunotoxina 192IgG-saporina.