The interaction of the Chrna5 D398N variant with developmental nicotine exposure.

A single nucleotide polymorphism (SNP) in CHRNA5 (rs16969968, change from an aspartic acid [D] to asparagine [N] at position 398 of the human α5 nicotinic acetylcholine receptor subunit) has been associated with increased risk for nicotine dependence. Consequently, carriers of the risk variant may be at elevated risk for in utero nicotine exposure. To assess whether this gene-environment interaction might impact nicotine intake in developmental nicotine-exposed offspring, we utilized a mouse expressing this human SNP. D and N dams drank nicotine (100 µg/mL) in 0.2% saccharin water or 0.2% saccharin water alone (vehicle) as their sole source of fluid from 30 days prior to breeding until weaning of offspring. The nicotine (D Nic, N Nic) or vehicle (D Veh, N Veh) exposed offspring underwent a 2-bottle choice test between postnatal ages of 30 to 46 days. N Nic offspring consumed the most nicotine at the highest concentration (400 µg/mL) compared with all other groups. In contrast, D Nic offspring drank the least amount of nicotine at all concentrations tested. Nicotine-stimulated dopamine (DA) release measured from striatal synaptosomes was increased in D Nic offspring, while decreased in N Nic offspring relative to their genotype-matched controls. These data suggest that the α5 variant influences the effect of developmental nicotine exposure on nicotine intake of exposed offspring. This gene-environment interaction on striatal DA release may provide motivation for increased nicotine seeking in N Nic offspring and reduced consumption in D Nic offspring.

In vivo chronic nicotine exposure differentially and reversibly affects upregulation and stoichiometry of α4ß2 nicotinic receptors in cortex and thalamus.

Studies with heterologous expression systems have shown that the α4ß2 nicotinic acetylcholine receptor (nAChR) subtype can exist in two stoichiometries (with two [(α4)2(ß2)3] or three [(α4)3(ß2)2] copies of the α subunit in the receptor pentamer) which have different pharmacological and functional properties and are differently regulated by chronic nicotine treatment. However, the effects of nicotine treatment in vivo on native α4ß2 nAChR stoichiometry are not well known. We investigated in C57BL/6 mice the in vivo effect of 14-day chronic nicotine treatment and subsequent withdrawal, on the subunit expression and ß2/α4 subunit ratio of (3)H-epibatidine labeled α4ß2*-nAChR in total homogenates of cortex and thalamus. We found that in basal conditions the ratio of the ß2/α4 subunit in the cortex and thalamus is different indicating a higher proportion in receptors with (α4)2(ß2)3 subunit stoichiometry in the thalamus. For cortex exposure to chronic nicotine elicited an increase in receptor density measured by (3)H-epibatidine binding, an increase in the α4 and ß2 protein levels, and an increase in ß2/α4 subunit ratio, that indicates an increased proportion of receptors with the (α4)2(ß2)3 stoichiometry. For thalamus we did not find a significant increase in receptor density, α4 and ß2 protein levels, or changes in ß2/α4 subunit ratio. All the changes elicited by chronic nicotine in cortex were transient and returned to basal levels with an average half-life of 2.8 days following nicotine withdrawal. These data suggest that chronic nicotine exposure in vivo favors increased assembly of α4ß2 nAChR containing three ß2 subunits. A greater change in stoichiometry was observed for cortex (which has relatively low basal expression of (α4)2(ß2)3 nAChR) than in thalamus (which has a relatively high basal expression of (α4)2(ß2)3 nAChR).

Nicotinic cholinergic mechanisms causing elevated dopamine release and abnormal locomotor behavior.

Firing rates of dopamine (DA) neurons in substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) control DA release in target structures such as striatum and prefrontal cortex. DA neuron firing in the soma and release probability at axon terminals are tightly regulated by cholinergic transmission and nicotinic acetylcholine receptors (nAChRs). To understand the role of α6* nAChRs in DA transmission, we studied several strains of mice expressing differing levels of mutant, hypersensitive (leucine 9' to serine [L9'S]) α6 subunits. α6 L9'S mice harboring six or more copies of the hypersensitive α6 gene exhibited spontaneous home-cage hyperactivity and novelty-induced locomotor activity, whereas mice with an equal number of WT and L9'S α6 genes had locomotor activity resembling that of control mice. α6-dependent, nicotine-stimulated locomotor activation was also more robust in high-copy α6 L9'S mice versus low-copy mice. In wheel-running experiments, results were also bi-modal; high-copy α6 L9'S animals exhibited blunted total wheel rotations during each day of a 9-day experiment, but low-copy α6 L9'S mice ran normally on the wheel. Reduced wheel running in hyperactive strains of α6 L9'S mice was attributable to a reduction in both overall running time and velocity. ACh and nicotine-stimulated DA release from striatal synaptosomes in α6 L9'S mice was well-correlated with behavioral phenotypes, supporting the hypothesis that augmented DA release mediates the altered behavior of α6 L9'S mice. This study highlights the precise control that the nicotinic cholinergic system exerts on DA transmission and provides further insights into the mechanisms and consequences of enhanced DA release.

Role of alpha5 nicotinic acetylcholine receptors in pharmacological and behavioral effects of nicotine in mice.

Incorporation of the alpha5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the alpha5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the alpha5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the alpha5 nAChR subunit in nicotine dependence. Using alpha5(-/-) mice, the current study aimed to examine the role of alpha5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. (86)Rb(+) efflux and (125)I-epibatidine binding assays were conducted to examine the effect of alpha5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that alpha5(-/-) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the alpha5 nAChR is involved in nicotine reward. Alternatively, alpha5(-/-) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the alpha5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that alpha5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence.

In vivo effects of 3-iodocytisine: pharmacological and genetic analysis of hypothermia and evaluation of chronic treatment on nicotinic binding sites.

Several cytisine derivatives have been developed in the search for more selective drugs at nicotinic acetylcholine receptors (nAChR). Binding experiments in transfected cell lines showed that the iodination of cytisine in the position 3 of the pyridone ring increased potency at alpha7-nAChR and to a lesser extent at the alpha4beta2 subtypes, both of which are widely expressed in the brain. However, no in vivo studies have been published on this compound. Inhibition curves presented here using wild type, beta2, and beta4-null mutant mice confirm that 3-IC binds to alpha4beta2 *, alpha7 * and alpha3beta4 * receptors with higher affinity than cytisine (asterisk indicates the receptor may contain additional subunits, Lukas et al., 1999). Intraperitoneal injection of 3-iodocytisine (3-IC) induced considerable dose-dependent hypothermia in DBA/2J and C57BL/6J mice. This response was blocked by mecamylamine and partially inhibited by hexamethonium. beta4-null mice displayed significantly less 3-IC-induced hypothermia than wild-type mice, beta2-null mice were somewhat less affected than wild types, while responses of alpha7 *-null mice were similar to wild types. Mice treated chronically with 3-IC display a marked increase in alpha7 * and alpha4beta2 * binding sites determined by radioligand binding in membrane preparations from cerebral cortex and hippocampus. Quantitative autoradiographic analysis of 28 brain regions of mice treated with 3-IC was consistent with the membrane binding, detecting an increase of cytisine-sensitive [(125)I]epibatidine binding sites, while cytisine-resistant [(125)I]epibatidine sites were unchanged. [(125)I]alpha-Bungarotoxin binding sites also exhibited up-regulation. These results give a first evaluation of in vivo consequences of 3-IC as a potent agonist with marked effects on mice.

Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation.

Genetic approaches identify differential roles for alpha4beta2* nicotinic receptors in acute models of antinociception in mice.

The effects of nicotine on the tail-flick and hot-plate tests were determined to identify nicotinic receptor subtypes responsible for spinally and supraspinally mediated nicotine analgesia in knockin mice expressing hypersensitive alpha(4) nicotinic receptors (L9'S), in seven inbred mouse strains (C57BL/6, DBA/2, A/2, CBA/2, BALB/cByJ, C3H/HeJ, and 129/SvEv), and in two F1 hybrids (B6CBAF1 and B6D2F1). L9'S heterozygotes were approximately 6-fold more sensitive to the antinociceptive effects of nicotine than the wild-type controls in the hot-plate test but not in the tail-flick assay. Large differences in the effects of nicotine were also observed with both tests for the seven mouse strains. A/J and 129 mice were 6- to 8-fold more sensitive than CBA and BALB mice. In addition, B6CBAF1 hybrid mice were even less sensitive than CBA mice. Nicotinic binding sites were measured in three spinal cord regions and the hindbrain of the inbred strains. Significant differences in cytisine-sensitive, high affinity [(125)I]epibatidine binding site levels (alpha(4)beta(2)(*) subtypes), but not in (125)I-alpha-bungarotoxin binding (alpha(7)(*) subtypes), were observed. Significant negative correlations between cytisine-sensitive [(125)I]epibatidine binding and nicotine ED(50) for both tests were noted. Our results indicate that alpha(4)beta(2)(*) acetylcholine nicotinic receptors (nAChR) are important in mediating nicotine analgesia in supraspinal responses, while also showing that alpha(4)beta(2)(*)-nAChR and at least one other nAChR subtype appear to modulate spinal actions.

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.

Nicotinic-agonist stimulated (86)Rb(+) efflux and [(3)H]epibatidine binding of mice differing in beta2 genotype.

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in beta2 subunit expression. Function was measured by on-line detection of (86)Rb(+) efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 microM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-beta-erythroidine (DHbetaE); and (2) stimulation with 10 microM epibatidine in the presence of 2 microM DHbetaE, a response that is relatively resistant to inhibition by DHbetaE. Deletion of the beta2 subunit profoundly reduced both DHbetaE-sensitive and -resistant (86)Rb(+) efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [(3)H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the beta2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in beta2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [(3)H]epibatidine binding was heterogeneous in that a fraction of the sites required the beta2 subunit. Many remaining sites were sensitive to inhibition by alpha-bungarotoxin indicating that a subset of the low affinity [(3)H]epibatidine binding are of the alpha7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for beta2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.

Identification of a novel nicotinic binding site in mouse brain using [(125)I]-epibatidine.

[(125)I]-Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high affinity. In this study, [(125)I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. Binding of [(125)I]-epibatidine was saturable and apparently monophasic in each brain region (K:(D:)=71+/-12 pM mean+/-s.e.mean across regions) but inhibition of [(125)I]-epibatidine binding (200 pM) by A85380, cytisine and (-)-nicotine was biphasic, indicating the presence of multiple binding sites. The sites with lower agonist affinity comprised 30.0+/-2.2, 58.6+/-0.1 and 48.7+/-3.3% of specific [(125)I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. The affinity difference between A85380-sensitive and -resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to differentiate agonist-sensitive and -resistant sites. The pharmacological profiles of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and five antagonists. The profiles were indistinguishable across regions, implying that A85380-resistant [(125)I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. The pharmacological profile of the A85380-resistant sites is very different from that previously reported for high affinity (-)-[(3)H]-nicotine-, [(125)I]-alpha-bungarotoxin-, or [(125)I]-alpha-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.

Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia.

Previous studies have suggested that an abnormality in neuronal nicotinic acetylcholine receptor expression or function may be involved in the neuropathophysiology of schizophrenia. [(3)H]-nicotine and [(3)H]-epibatidine binding were compared in postmortem brain from control and schizophrenic subjects with varying smoking histories. In control subjects, increased receptor binding was seen in hippocampus, cortex, and caudate with increasing tobacco use. In contrast, schizophrenic smokers had reduced nicotinic receptor levels in these brain regions compared to control smokers. Chronic haloperidol and nicotine treatment, in the rat, was used to assess neuroleptic effects on receptor up-regulation by nicotine. A significant increase in cortical nicotinic receptors was seen in both nicotine treated as well as haloperidol and nicotine co-treated animals, suggesting that the abnormal regulation of high affinity neuronal nicotinic receptors in schizophrenics following nicotine use was not related to chronic neuroleptic treatment.

125I-alpha-conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain.

alpha-Conotoxin MII (CtxMII), a peptide toxin from the venom of the predatory cone snail Conus magus, displays an unusual nicotinic pharmacology. Specific binding of a radioiodinated derivative ((125)I-alpha-CtxMII) was identified in brain region homogenates and tissue sections. Quantitative autoradiography indicated that (125)I-alpha-CtxMII binding sites have an unique pharmacological profile and distribution in mouse brain, being largely confined to the superficial layers of the superior colliculus, nigrostriatal pathway, optic tract, olivary pretectal, and mediolateral and dorsolateral geniculate nuclei. Expression of alpha-CtxMII binding sites in the nigrostriatal pathway, combined with evidence for alpha-CtxMII-sensitivity of nicotine-induced [(3)H]dopamine release in rodent striatal preparations indicates that (125)I-alpha-CtxMII binding nicotinic acetylcholine receptors are likely to be physiologically important. Unlabeled alpha-CtxMII potently (K(i) < 3 nM) competed for a subset of [(3)H]epibatidine binding sites in mouse brain homogenates, but weakly (IC(50) > 10 microM) interacted with (125)I-alpha-bungarotoxin and (-)-[(3)H]nicotine binding sites, confirming this compound's novel nicotinic pharmacology. Quantitative autoradiography revealed that alpha-CtxMII binds with high affinity at a subset of [(3)H]epibatidine binding sites with relatively low cytisine affinity ("cytisine-resistant" sites), resolving [(3)H]epibatidine binding into three different populations, each probably corresponding to a receptor subtype. The majority population seems to correspond to that which binds nicotine and cytisine with high affinity ("cytisine-sensitive" sites). Comparison of the cytisine-resistant population's distribution with that of alpha3 subunit mRNA expression suggests that the fractions both more and less sensitive to alpha-CtxMII probably contain the alpha3 subunit, perhaps in combination with different beta subunits.

Pharmacological and null mutation approaches reveal nicotinic receptor diversity.

We have developed an array of assays for nicotinic acetylcholine receptor binding and function. [125I]alpha-Bungarotoxin-, (-)-[3H]nicotine-, and [3H]epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic acetylcholine receptor function was quantified using synaptosomal [3H]dopamine, [3H]gamma-aminobutyric acid ([3H]GABA), and 86Rb(+) efflux techniques. Additionally, the effects of beta2 subunit deletion on each of the measures were assessed. Detailed pharmacological comparison revealed minimally six nicotinic binding subtypes: [125I]alpha-bungarotoxin-binding nicotinic acetylcholine receptors; beta2-subunit-dependent and -independent high-affinity (-)-[3H]nicotine-binding sites; beta2-dependent and -independent cytisine-resistant [3H]epibatidine-binding sites; and a beta2-dependent low-affinity [3H]epibatidine binding site. Comparative pharmacology suggested that [3H]GABA and dihydro-beta-erythroidine (DHbetaE)-sensitive 86Rb(+) efflux are mediated by the same (probably alpha4beta2) nicotinic acetylcholine receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke [3H]dopamine and DHbetaE-resistant 86Rb(+) efflux. In whole-brain preparations, each measure of nicotinic acetylcholine receptor function was beta2 dependent. The majority of beta2-independent [3H]epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification of novel, native nicotinic acetylcholine receptors.

Potential role of the alpha4 and alpha6 nicotinic receptor subunits in regulating nicotine-induced seizures.

Several studies have shown that genetic factors influence sensitivity to nicotine-induced seizures in the mouse. We used recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines to assess the possibility that polymorphisms associated with one or more of the nicotinic receptors cosegregate with differential sensitivity to nicotine-induced seizures. Restriction fragment length polymorphisms (RFLPs) associated with the alpha2, alpha3, alpha4, alpha5, and alpha6 nicotinic receptors were identified in the LS and SS mouse lines, but the RI strains were polymorphic for only the alpha4 and alpha6 RFLPs. The RI strains were tested for sensitivity to nicotine-induced seizures. Strain and gender effects on seizure sensitivity were obtained as assessed by ED(50) values and latency to seizure. Those RI strains with the LS-like alpha4 RFLP were, on average, more sensitive to nicotine-induced seizures than were those RI strains with SS-like alpha4 RFLP. The alpha6 nicotine receptor may also play a role in modulating nicotine-induced seizures, but this effect is markedly influenced by gender. Females of the RI strains with the LS-like alpha6 RFLP were more sensitive to nicotine than were females of the strains with the SS-like alpha6 RFLP. Similar trends were seen in the males, but these trends were not significant. Thus, these strain differences may be due to polymorphisms associated with both the alpha4 and alpha6 nicotinic receptors, but gender also plays an important role in regulating sensitivity to nicotine-induced seizure.

UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

Desensitization of nicotinic agonist-induced [3H]gamma-aminobutyric acid release from mouse brain synaptosomes is produced by subactivating concentrations of agonists.

Several neurochemical and electrophysiological studies have shown that neuronal nicotinic receptors are desensitized by pretreatment with lower agonist concentrations than are required to activate the receptors, but the extent of desensitization and agonist concentration required to produce desensitization vary depending upon receptor subtype. Recently, we reported that nicotinic agonists will stimulate the release of [3H]gamma-aminobutyric acid (GABA) from synaptosomes prepared from mouse brain. The studies described herein evaluated desensitization of [3H]GABA release produced by pretreatment with 12 nicotinic agonists. Pretreatment produced near total desensitization that developed slowly (onset T(1/2) = 3.46 min) and was totally reversible (recovery T(1/2) = 4.95 min). Nine of the 12 compounds tested induced total or near total desensitization at concentrations that were less than those required to produce a reliably measured increase in [3H]GABA release. Nicotine produced total block with an IC(50) value of 26 nM. This value is two orders of magnitude lower than the EC(50) for nicotine-induced [3H]GABA release (1630 nM). The three compounds that showed an overlap of the desensitization and activation concentration-effect curves (cytisine, anabasine, nornicotine) are all partial agonists. Comparison of the desensitization properties of the [3H]GABA release with an ion ((86)Rb+) efflux that we have measured previously suggests that the receptor that mediates GABA release and (86)Rb(+) efflux is the same, most likely the alpha4beta2 subtype.

An autoradiographic study of the distribution of binding sites for the novel alpha7-selective nicotinic radioligand [3H]-methyllycaconitine in the mouse brain.

[3H]-Methyllycaconitine ([3H]-MLA) is a new radioligand with selectivity for alpha7-type neuronal nicotinic acetylcholine receptors (nAChRs). In our previous study [Davies, A.R.L., Hardick, D.J., Blagbrough, I.S., Potter, B.V.L., Wolstenholme, A.J. & Wonnacott, S. (1999) Neuropharmacology, 38, 679-690], this radioligand labelled a single class of site in rat brain membranes; its pharmacology and distribution in crudely dissected brain regions closely paralleled that of the well-established alpha7-ligand [125I]-alpha-bungarotoxin. However, a small population of [3H]-MLA binding sites was apparently insensitive to alpha-bungarotoxin. Here we have extended the study to mouse brain, using autoradiography to examine the distribution of [3H]-MLA and [125I]-alpha-bungarotoxin binding sites. [3H]-MLA labelled a single class of site in mouse brain membranes with a KD of 2.2 nM and a Bmax of 45.6 fmol/mg protein. Specific binding, defined by unlabelled MLA (Ki = 0.69 nM), was completely inhibited by (-)-nicotine (Ki = 1.62 microM), whereas alpha-bungarotoxin inhibited only 85% of specific binding (Ki = 3.5 nM). The distributions of [125I]-alpha-bungarotoxin and [3H]-MLA binding sites were compared by autoradiography, and binding was quantitated in 72 brain regions. Binding of both radioligands was highly correlated, with highest densities in the dorsal tegmental nucleus of the pons, colliculi and hippocampus. Serial sections labelled with [3H]-MLA in the absence or presence of unlabelled MLA or alpha-bungarotoxin provided no evidence for any alpha-bungarotoxin-resistant binding. The results are discussed in terms of binding sites that are inaccessible to alpha-bungarotoxin in membrane preparations. This study demonstrates the utility of [3H]-MLA for characterization of alpha7-type nicotinic receptors in mammalian brain, and suggests that it labels a population identical to that defined by [125I]-alpha-bungarotoxin.

Two pharmacologically distinct components of nicotinic receptor-mediated rubidium efflux in mouse brain require the beta2 subunit.

Nicotinic agonist-stimulated efflux of 86Rb+ from mouse brain synaptosomes was monitored continuously by on-line radioactivity detection. The concentration-effect curve following a 5-s stimulation with acetylcholine was biphasic (EC50 = 7.2 and 550 microM). alpha-Bungarotoxin (100 nM) did not inhibit the response, but dihydro-beta-erythroidine (DHbetaE) blocked both phases with differing potency (average IC50 =.22 and 8.9 microM for responses activated by low and high acetylcholine concentrations, respectively). Differential sensitivity DHbetaE inhibition was used to measure stimulation of 86Rb+ efflux by 17 nicotinic agonists, which differed markedly in potency and efficacy. All agonists were more potent at the DHbetaE-sensitive site. Both components were inhibited by the six antagonists tested. Methyllycaconitine and DHbetaE were more potent for the DHbetaE-sensitive component, whereas hexamethonium was more potent at the DHbetaE-resistant component. Both DHbetaE-sensitive and DHbetaE-resistant responses were reduced more than 95% in beta2-null mutant mice, establishing the requirement for the beta2 subunit for both components. Both components were widely, but not identically, distributed throughout the brain. The DHbetaE-sensitive component appears to be identical with agonist-stimulated 86Rb+ efflux described previously and is likely to be mediated by alpha4beta2 receptors. The DHbetaE-resistant component is a novel, active, and widely distributed response mediated by nicotinic receptor(s) that also require the beta2 subunit.