Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment

Chronic nicotine exposure up-regulates neuronal nicotinic receptors, but the functional consequences for these receptors is less well understood. Following 2 weeks of nicotine or saline treatment by osmotic minipump, the functional activity of nicotinic receptors was measured by concentration-response curves for epibatidine-stimulated (86)Rb efflux. Nicotine-treated animals had a significantly higher maximal efflux in cerebral cortex and superior colliculus, but not in thalamus or interpeduncular nucleus plus medial habenula. This increase was confirmed in a separate experiment with stimulation by single concentrations of epibatidine (cortex, superior colliculus) or nicotine (cortex only). Chronic nicotine did not alter (86)Rb efflux stimulated by cytisine, an alpha3beta4-selective agonist, or by potassium chloride, in any region. Short-term (16 h) nicotine exposure caused no changes in either (86)Rb efflux or receptor binding measured with [(3)H]epibatidine. Binding was significantly increased after 2 weeks nicotine exposure in cortex, superior colliculus and thalamus, but not in interpeduncular nucleus plus medial habenula. The increases in epibatidine-stimulated (86)Rb efflux in the four regions tested was linearly correlated with the increases in [(3)H]epibatidine binding in these regions (R(2) = 0.91), suggesting that rat brain receptors up-regulated by chronic nicotine are active. These results have important consequences for understanding nicotinic receptor neurobiology in smokers and users of nicotine replacement therapy.     

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.     

bis-Azaaromatic quaternary ammonium analogues: ligands for alpha4beta2* and alpha7* subtypes of neuronal nicotinic receptors

A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N,N'-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [(3)H]nicotine binding sites (K(i)=330 nM), but did not inhibit [(3)H]methyllycaconitine binding (K(i) >100 microM), indicative of an interaction with alpha4beta2*, but not alpha7* receptor subtypes, respectively. Also, bNDI inhibited (IC(50)=3.76 microM) nicotine-evoked (86)Rb(+) efflux from rat thalamic synaptosomes, indicating antagonist activity at alpha4beta2* nAChRs. N,N'-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [(3)H]methyllycaconitine binding sites (K(i)=1.61 microM), but did not inhibit [(3)H]nicotine binding (K(i)>100 microM), demonstrating an interaction with alpha7*, but not alpha4beta2* nAChRs. Thus, variation of N-n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the alpha4beta2* nAChR subtype, as well as ligands with selectivity at alpha7* nAChRs.     

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.     

Autoradiographic distribution of high affinity muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine in rat brain

The relative distribution of muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine was determined using autoradiography. [3H]Acetylcholine binding to high affinity muscarinic receptors was similar to what has been described for an M-2 distribution: highest levels of binding occurred in the pontine and brainstem nuclei, anterior pretectal area and anteroventral thalamic nucleus, while lower levels occurred in the caudate-putamen, accumbens nucleus and primary olfactory cortex. Nicotinic receptors were labeled with [3H]acetylcholine to the greatest extent in the interpeduncular nucleus, several thalamic nuclei, medial habenula, presubiculum and superior colliculus, and to the least extent in the hippocampus and inferior colliculus. By using autoradiography to localize cholinergic binding sites throughout the brain it was observed that the distributions of high affinity muscarinic and nicotinic sites labeled with the endogenous ligand, [3H]acetylcholine are different from each other and are different from distributions of muscarinic and nicotinic sites labeled with muscarinic and nicotinic antagonists.     

Autoradiographic localization of nicotinic receptor binding in rat brain using [3H]methylcarbamylcholine, a novel radioligand

Light microscopic autoradiography was used to visualize the neuroanatomical distribution of nicotinic receptors in rat brain using a novel radioligand, [3H]methylcarbamylcholine (MCC). Specific [3H]MCC binding to slide-mounted tissue sections of rat brain was saturable, reversible and of high affinity. Data analysis revealed a single population of [3H]MCC binding sites with a Kd value of 1.8 nM and Bmax of 20.1 fmol/mg protein. Nicotinic agonists and antagonists competed for [3H]MCC binding sites in slide-mounted brain sections with much greater potency than muscarinic drugs. The rat brain areas containing the highest densities of [3H]MCC binding were in thalamic regions, the medial habenular nucleus and the superior colliculus. Moderate densities of [3H]MCC binding were seen over the anterior cingulate cortex, the nucleus accumbens, the zona compacta of substantia nigra and ventral tegmental area. Low densities of [3H]MCC binding were found in most other brain regions. These data suggest that [3H]MCC selectively labels central nicotinic receptors and that these receptors are concentrated in the thalamus, the medial habenular nucleus and the superior colliculus of the rat brain.     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.     

Biochemical Characterization of the Nicotinic Cholinergic Receptors in Human Brain: Binding of (—)-[3H]Nicotine

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

Nicotinic activation of mesolimbic neurons assessed by rubidium efflux in rat accumbens and ventral tegmentum

Dopaminergic mesolimbic neurons, with cell bodies in the ventral tegmental area (VTA) projecting to the nucleus accumbens (NAc), have been shown to be involved in the development of drug dependence. The application of nicotine to either the VTA or NAc produces an increase in dopamine release; however, the positive reinforcement produced by the systemic injection of nicotine is primarily due to stimulation of nicotinic acetylcholine receptors (nAChRs) in the VTA. Because the brain levels of nicotine would likely be the same in both brain areas, the nAChRs in the NAc may be less sensitive than those in the VTA. This study was undertaken to make a direct comparison of the native nAChRs in intact slices of NAc and VTA by measuring nicotine-stimulated efflux of (86)Rb(+) in a superfusion assay. The potency of nicotine and several other agonists was similar in both brain areas, but nicotine was somewhat more efficacious in the NAc. The effects of treatment duration, calcium and nicotinic antagonists were also determined. The results suggest that the predominant effect of nicotine in the VTA following systemic administration is due to differences in neuronal circuitry or firing patterns rather than inherent differences in the two nAChR populations.     

Synthesis, nicotinic acetylcholine receptor binding, and pharmacological properties of 3'-(substituted phenyl)deschloroepibatidine analogs

A series of 3'-(substituted phenyl)deschloroepibatidine analogs (5a-j) were synthesized. The alpha4beta2( *) and alpha7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a-j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13), the partial agonist, varenicline (3), and the antagonist 2'-fluoro-3'-(substituted phenyl)deschloroepibatidine analogs (7a-j). Unlike epibatidine and deschloroepibatidine, which are potent agonists in the tail-flick test, 5a-k show no or very low antinociceptive activity in the tail-flick or hot-plate test. However, they are potent antagonists in nicotine-induced antinociception in the tail-flick test, but weaker than the corresponding 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

Synthesis and radiosynthesis of [(18)F]FPhEP, a novel alpha(4)beta(2)-selective, epibatidine-based antagonist for PET imaging of nicotinic acetylcholine receptors

FPhEP (1, (+/-)-2-exo-(2'-fluoro-3'-phenyl-pyridin-5'-yl)-7-azabicyclo[2.2.1]heptane) belongs to a recently described novel series of 3'-phenyl analogues of epibatidine, which not only possess subnanomolar affinity and high selectivity for brain alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs), but also were reported as functional antagonists of low toxicity (up to 15 mg/kg in mice). FPhEP (1, K(i) of 0.24 nM against [(3)H]epibatidine) as reference as well as the corresponding N-Boc-protected chloro- and bromo derivatives (3a,b) as precursors for labelling with fluorine-18 were synthesized in eight and nine steps, respectively, from commercially available N-Boc-pyrrole (overall yields=17% for 1, 9% for 3a and 8% for 3b). FPhEP (1) was labelled with fluorine-18 using the following two-step radiochemical process: (1) no-carrier-added nucleophilic heteroaromatic ortho-radiofluorination from the corresponding N-Boc-protected chloro- or bromo derivatives (3 a,b-1mg) and the activated K[(18)F]F-Kryptofix(222) complex in DMSO using microwave activation at 250 W for 1.5 min, followed by (2) quantitative TFA-induced removal of the N-Boc-protective group. Radiochemically pure (>99%) [(18)F]FPhEP ([(18)F]-1, 2.22-3.33 GBq, 66-137 GBq/micromol) was obtained after semi-preparative HPLC (Symmetry C18, eluent aq 0.05 M NaH(2)PO(4)/CH(3)CN, 80:20 (v:v)) in 75-80 min starting from a 18.5 GBq aliquot of a cyclotron-produced [(18)F]fluoride production batch (10-20% nondecay-corrected overall yield). In vitro binding studies on rat whole-brain membranes demonstrated a subnanomolar affinity (K(D) 660 pM) of [(18)F]FPhEP ([(18)F]-1) for nAChRs. In vitro autoradiographic studies also showed a good contrast between nAChR-rich and -poor regions with a low non-specific binding. Comparison of in vivo Positron Emission Tomography (PET) kinetics of [(18)F]FPhEP ([(18)F]-1) and [(18)F]F-A-85380 in baboons demonstrated faster brain kinetics of the former compound (with a peak uptake at 20 min post injection only). Taken together, the preliminary data obtained confirm that [(18)F]FPhEP ([(18)F]-1) has potential for in vivo imaging nAChRs in the brain with PET.     

Phosphorimaging detection and quantitation for isotopic ion flux assays

A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated (22)Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated (86)Rb efflux produced data similar to data obtained with the (22)Na influx assay. However, assays for (45)Ca influx were not successful, although (45)Ca was readily detected and quantified. Voltage-gated sodium channel-mediated (22)Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1,200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.     

The synthesis and nicotinic binding activity of (+/-)-epiquinamide and (+/-)-C(1)-epiepiquinamide

The synthesis of (+/-)-epiquinamide 1 and (+/-)-C(1)-epiepiquinamide 2 based on the use of a Curtius rearrangement to introduce the C(1) amino residue is reported. In a competition binding assay for [(3)H]epibatidine binding to rat brain membranes neither (+/-)-1 nor (+/-)-2 showed any significant level of nicotinic activity.     

Synthesis and pharmacological characterization of bivalent ligands of epibatidine at neuronal nicotinic acetylcholine receptors

A series of bivalent ligands 6a-d of epibatidine were synthesized. All four ligands showed nanomolar binding affinities at six neuronal nicotinic acetylcholine receptor (nAChR) subtypes in competition binding assays. In contrast to epibatidine, these bivalent ligands are weak partial agonists at the alpha3beta4 nAChR as shown by functional assays.     

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Different oxidative profile and nicotinic receptor interaction of amphetamine and 3,4-methylenedioxy-methamphetamine

d-Amphetamine (AMPH) and MDMA increased intracellular production of reactive oxygen species (ROS) in isolated mouse striatal synaptosomes. MDMA showed a maximal oxidative effect at 50-100 microM. However, for AMPH a double maximum was obtained, the first between 0.1 and 1 microM and the second at 1mM. No oxidative effect was present in synaptosomes from reserpinized mice. Cocaine and l-deprenyl inhibited MDMA and AMPH (0.1 microM) ROS production but not that of AMPH at a higher concentration (1mM). When this high concentration was used, its oxidative effect was abolished by a phospholipase A(2) inhibitor. Delta(9)-Tetrahydrocannabinol fully prevented the oxidative effect of AMPH and MDMA, by a CB(1) receptor-independent mechanism, as did it NPC 15437 and genistein. The pro-oxidative effect induced by AMPH and MDMA showed a strong dependence on calcium (extracellular and from internal stores) and also was inhibited by nicotinic receptor (nAChR) antagonists dihydro-beta-erythroidine, methyllycaconitine (MLA) and alpha-bungarotoxin. MDMA displaced [(3)H]epibatidine and [(3)H]MLA binding with higher affinity than AMPH. Both amphetamines competitively displaced [(3)H]epibatidine from heteromeric receptors but results obtained from [(3)H]MLA binding demonstrated a non-competitive profile. Preincubation of PC12 cells with AMPH or MDMA reduced [(3)H]dopamine uptake. For MDMA, this effect was prevented by MLA. To summarize, comparing AMPH and MDMA we have demonstrated that these drugs induce an oxidative effect dependent on drug concentration and also reduce dopamine uptake. Processes that are known to affect dopamine transporter functionality also seem to modulate amphetamine derivatives-induced ROS production. For MDMA, acute effects tested are blocked by nAChR antagonists, which points to the possibility that these antagonists could be used to treat some of the adverse effects described in MDMA abusers. Conversely, no implication of nicotinic receptors has been proved for AMPH-induced effects at concentrations achievable in CNS after its administration.