Manolo Mugnaini

Nicotinic Acetylcholine Receptors in the Mesolimbic Pathway: Primary Role of Ventral Tegmental Area α6β2* Receptors in Mediating Systemic Nicotine Effects on Dopamine Release, Locomotion, and Reinforcement

α6* nicotinic acetylcholine receptors (nAChRs) are highly and selectively expressed by mesostriatal dopamine (DA) neurons. These neurons are thought to mediate several behavioral effects of nicotine, including locomotion, habit learning, and reinforcement. Yet the functional role of α6* nAChRs in midbrain DA neurons is mostly unknown. The aim of this study was to determine the composition and in vivo functional role of α6* nAChR in mesolimbic DA neurons of male rats. Immunoprecipitation and immunopurification techniques coupled with cell-specific lesions showed that the composition of α6* nAChR in the mesostriatal system is heterogeneous, with (non-α4)α6β2* being predominant in the mesolimbic pathway and α4α6β2* in the nigrostriatal pathway. We verified whether α6* receptors mediate the systemic effects of nicotine on the mesolimbic DA pathway by perfusing the selective antagonists α-conotoxin MII (CntxMII) (α3/α6β2* selective) or α-conotoxin PIA (CntxPIA) (α6β2* selective) into ventral tegmental area (VTA). The intra-VTA perfusion of CntxMII or CntxPIA markedly decreased systemic nicotine-elicited DA release in the nucleus accumbens and habituated locomotion; the intra-VTA perfusion of CntxMII also decreased the rate of nicotine infusion in the maintenance phase of nicotine, but not of food, self-administration. Overall, the results of these experiments show that the α6β2* nAChRs expressed in the VTA are necessary for the effects of systemic nicotine on DA neuron activity and DA-dependent behaviors such as locomotion and reinforcement, and suggest that α6β2*-selective compounds capable of crossing the blood–brain barrier may affect the addictive properties of nicotine and therefore be useful in the treatment of tobacco dependence.

The Use of Knock-Out Mice Unravels Distinct Roles for mGlu2 and mGlu3 Metabotropic Glutamate Receptors in Mechanisms of Neurodegeneration/Neuroprotection

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (−)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2−/−, or mGlu3−/− mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2−/− mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2−/− mice but not in mGlu3−/− mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

Imaging Dopamine D3 Receptors in the Human Brain with Positron Emission Tomography, [11C]PHNO, and a Selective D3 Receptor Antagonist

1. Clinical Imaging Centre, GlaxoSmithKline, London, U.K. 2. Neurosciences Centre of Excellence in Drug Discovery, GlaxoSmithKline, Verona, Italy 3. Department of Psychiatry, Columbia University, New York, USA 4. Centre for Addiction and Mental Health, Toronto, Canada 5. Department of Engineering Science, University of Oxford, U.K. 6. Division of Neuroscience and Mental Health, Imperial College, London, U.K.BACKGROUND Dopamine D(3) receptors are involved in the pathophysiology of several neuropsychiatric conditions. [(11)C]-(+)-PHNO is a radiolabeled D(2) and D(3) agonist, suitable for imaging the agonist binding sites (denoted D(2HIGH) and D(3)) of these receptors with positron emission tomography (PET). PET studies in nonhuman primates documented that, in vivo, [(11)C]-(+)-PHNO displays a relative selectivity for D(3) compared with D(2HIGH) receptor sites and that the [(11)C]-(+)-PHNO signal is enriched in D(3) contribution compared with conventional ligands such as [(11)C] raclopride. METHODS To define the D(3) contribution (f(PHNO)(D3)) to [(11)C]-(+)-PHNO binding potential (BP(ND)) in healthy humans, 52 PET scans were obtained in 19 healthy volunteers at baseline and following oral administration of various doses of the selective D(3) receptor antagonist, GSK598809. RESULTS The impact of GSK598809 on [(11)C]-(+)-PHNO was regionally selective. In dorsal regions of the striatum, GSK598809 did not significantly affect [(11)C]-(+)-PHNO BP(ND) (f(PHNO)(D3) approximately 0%). Conversely, in the substantia nigra, GSK598809 dose-dependently reduced [(11)C]-(+)-PHNO binding to nonspecific level (f(PHNO)(D3) approximately 100%). In ventral striatum (VST), globus pallidus and thalamus (THA), [(11)C]-(+)-PHNO BP(ND) was attributable to a combination of D(2HIGH) and D(3) receptor sites, with f(PHNO)(D3) of 26%, 67% and 46%, respectively. D(3) receptor binding potential (BP(ND)(D3)) was highest in globus pallidus (1.90) and substantial nigra (1.39), with lower levels in VST (.77) and THA (.18) and negligible levels in dorsal striatum. CONCLUSIONS This study elucidated the pharmacologic nature of the [(11)C]-(+)-PHNO signal in healthy subjects and provided the first quantification of D(3) receptor availability with PET in the living human brain.

Upregulation of [3H]methyllycaconitine binding sites following continuous infusion of nicotine, without changes of α7 or α6 subunit mRNA: an autoradiography and in situ hybridization study in rat brain

It is well established that exposure of experimental animals to nicotine results in upregulation of the α4β2‐subtype of neuronal nicotinic acetylcholine receptors (nAChRs). The aim of this study was to determine the effect of nicotine on the levels of α7‐nAChRs in rat brain, for which only partial information is available. Rats were infused with nicotine (3 mg/kg/day) or saline for 2 weeks and their brains processed for receptor autoradiography with [3H]methyllycaconitine (MLA), a radioligand with nanomolar affinity for α7‐nAChRs. In control rats binding was high in hippocampus, intermediate in cerebral cortex and hypothalamus, and low in striatum, thalamus and cerebellum. There was high correlation between the distribution of [3H]MLA binding sites and α7 subunit mRNA (r = 0.816).

Selective down-regulation of [125i]Y0-α-conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine

Prolonged exposure to nicotine, as occurs in smokers, results in up-regulation of all the neuronal nicotinic acetylcholine receptor subtypes studied so far, the only differences residing in the extent and time course of the up-regulation. alpha6beta2*-Nicotinic acetylcholine receptors are selectively enriched in the mesostriatal dopaminergic system and may play a crucial role in nicotine dependence. Here we show that chronic nicotine treatment (3mg/kg/day for two weeks, via s.c. osmotic minipumps) caused a significant decrease (36% on average) in the binding of [(125)I]Y(0)-alpha-conotoxin MII (a selective ligand for alpha6beta2*-nicotinic acetylcholine receptors in this system) to all the five regions of the rat dopaminergic pathway analyzed in this study. After one week of withdrawal, binding was still lower than control in striatal terminal regions (namely the caudate putamen and the accumbens shell and core). In somatodendritic regions (the ventral tegmental area and the substantia nigra) the decrease was significant at the end of the treatment and recovered within one day of withdrawal. This effect was not due to displacement of [(125)I]Y(0)-alpha-conotoxin MII binding by residual nicotine. In fact the binding was not changed by 565 ng/g nicotine (obtained with a single injection of nicotine), a concentration much higher than that found in the brain of rats chronically treated with nicotine (240 ng/g). In addition, consistent with previous studies reporting an up-regulation of other subtypes of nicotinic acetylcholine receptors, we found that nicotine exposure significantly increased (40% on average) the binding of [(125)I]epibatidine (a non-selective agonist at most neuronal heteromeric nicotinic acetylcholine receptors) in three up to five regions containing only alpha-conotoxin MII-insensitive [(125)I]epibatidine binding sites, namely the primary motor, somatosensory and auditory cortices. In conclusion, this work is the first to demonstrate that alpha6beta2*-nicotinic acetylcholine receptors, unique within the nicotinic acetylcholine receptor family, are down-regulated following chronic nicotine treatment in rat dopaminergic mesostriatal pathway, a finding that may shed new light in the complex mechanisms of nicotine dependence.

Occupancy of Brain Dopamine D3 Receptors and Drug Craving: A Translational Approach

Selective dopamine D3 receptor (D3R) antagonists prevent reinstatement of drug-seeking behavior and decrease the rewarding effects of contextual cues associated with drug intake preclinically, suggesting that they may reduce drug craving in humans. GSK598809 is a selective D3R antagonist recently progressed in Phase I trials. The aim of this study was to establish a model, based on the determination of the occupancy of brain D3Rs (OD3R) across species, to predict the ability of GSK598809 to reduce nicotine-seeking behavior in humans, here assessed as cigarette craving in smokers. Using ex vivo [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([125I]7OH-PIPAT) autoradiography and [11C]PHNO positron emission tomography, we demonstrated a dose-dependent occupancy of the D3Rs by GSK598809 in rat, baboon, and human brains. We also showed a direct relationship between OD3R and pharmacokinetic exposure, and potencies in line with the in vitro binding affinity. Likewise, GSK598809 dose dependently reduced the expression of nicotine-induced conditioned place preference (CPP) in rats, with an effect proportional to the exposure and OD3R at every time point, and 100% effect at OD3R values ⩾72%. In humans, a single dose of GSK598809, giving submaximal levels (72–89%) of OD3R, transiently alleviated craving in smokers after overnight abstinence. These data suggest that either higher OD3R is required for a full effect in humans or that nicotine-seeking behavior in CPP rats only partially translates into craving for cigarettes in short-term abstinent smokers. In addition, they provide the first clinical evidence of potential efficacy of a selective D3R antagonist for the treatment of substance-use disorders.

A comparative study of the effects of the intravenous self-administration or subcutaneous minipump infusion of nicotine on the expression of brain neuronal nicotinic receptor subtypes

Long-term nicotine exposure changes neuronal acetylcholine nicotinic receptor (nAChR) subtype expression in the brains of smokers and experimental animals. The aim of this study was to investigate nicotine-induced changes in nAChR expression in two models commonly used to describe the effects of nicotine in animals: operant (two-lever presses) intravenous self-administration (SA) and passive subcutaneous nicotine administration via an osmotic minipump (MP). In the MP group, α4β2 nAChRs were up-regulated in all brain regions, α6β2* nAChRs were down-regulated in the nucleus accumbens (NAc) and caudate-putamen, and α7 nAChRs were up-regulated in the caudal cerebral cortex (CCx); the up-regulation of α4β2α5 nAChRs in the CCx was also suggested. In the SA group, α4β2 up-regulation was lower and limited to the CCx and NAc; there were no detectable changes in α6β2* or α7 nACRs. In the CCx of the MP rats, there was a close correlation between the increase in α4β2 binding and α4 and β2 subunit levels measured by means of Western blotting, demonstrating that the up-regulation was due to an increase in α4β2 proteins. Western blotting also showed that the increase in the β2 subunit exceeded that of the α4 subunit, suggesting that a change in α4β2 stoichiometry may occur in vivo as has been shown in vitro. These results show that nicotine has an area-specific effect on receptor subtypes, regardless of its administration route, but the effect is quantitatively greater in the case of MP administration.

1,2,4-Triazolyl Azabicyclo[3.1.0]hexanes: A New Series of Potent and Selective Dopamine D3 Receptor Antagonists

The discovery of new highly potent and selective dopamine (DA) D(3) receptor antagonists has recently allowed the characterization of the DA D(3) receptor in a range of preclinical animal models of drug addiction. A novel series of 1,2,4-triazol-3-yl-azabicyclo[3.1.0]hexanes, members of which showed a high affinity and selectivity for the DA D(3) receptor and excellent pharmacokinetic profiles, is reported here. Members of a group of derivatives from this series showed good oral bioavailability and brain penetration and very high in vitro affinity and selectivity for the DA D(3) receptor, as well as high in vitro potency for antagonism at this receptor. Several members of this series also significantly attenuate the expression of conditioned place preference (CPP) to nicotine and cocaine.

Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists

Despite growing evidence that inhibition of α6β2‐containing (α6β2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of α6β2*‐nAChRs has delayed the discovery of α6β2‐selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with α6β2 properties, to enable pharmacological characterization and the identification of novel α6β2‐selective antagonists.

Regionally different N‐methyl‐D‐aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]‐CGP 39653 in rat brain

1 Binding of D, L‐(E)‐2‐amino‐4‐[3H]‐propyl‐5‐phosphono‐3‐pentenoic acid ([3H]‐CGP 39653), a high affinity, selective antagonist at the glutamate site of the N‐methyl‐D‐aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2 [3H]‐CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg−1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3 Displacement analyses of [3H]‐CGP 39653 in striatum and cerebellum, performed with L‐glutamic acid, 3‐((±)‐2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L‐Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]‐CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, oneway ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fishers protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4 Regional binding of [3H]‐CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabeled antagonists at the NMDA site (D‐2‐[3H]‐amino‐5‐phosphonopentanoic acid ([3H]‐D‐AP5) and [3H]‐CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5 [3H]‐CGP 39653 binding was inhibited by increasing concentrations of L‐glutamic acid, CPP and glycine. L‐Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 μm and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]‐CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two‐ways ANOVA). Multiple comparison, performed by Fishers protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6 The inhibitory action of 10 μm glycine was reversed by 100 μm 7‐chloro‐kynurenic acid (7‐CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7‐CKA was not the same in all regions (P < 0.05, two‐ways ANOVA). In fact, in the presence of 10 μm glycine and 100 μm 7‐KCA, specific [3H]‐CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fishers protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7 These results demonstrate that [3H]‐CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]‐CGP 39653 binding modulation through the associated glycine site.