Altered hippocampal circuit function in C3H alpha7 null mutant heterozygous mice.

The alpha7 subtype of nicotinic receptor is highly expressed in the hippocampus where it is purported to modulate release of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The alpha7 receptor-mediated release of GABA is thought to contribute to hippocampal inhibition (gating) of response to repetitive auditory stimulation. This hypothesis is supported by observations of hippocampal auditory gating deficits in mouse strains with low levels of hippocampal alpha7 receptors compared to strains with high levels of hippocampal alpha7 receptors. The difficulty with comparisons between mouse strains, however, is that different strains have different genetic backgrounds. Thus, the observed interstrain differences in hippocampal auditory gating might result from factors other than interstrain variations in the density of hippocampal alpha7 receptors. To address this issue, hippocampal binding of the alpha7 receptor-selective antagonist alpha-bungarotoxin as well as hippocampal auditory gating characteristics were compared in C3H wild type and C3H alpha7 receptor null mutant heterozygous mice. The C3H alpha7 heterozygous mice exhibited significant reductions in hippocampal alpha7 receptor levels and abnormal hippocampal auditory gating compared to the C3H wild type mice. In addition, a general increase in CA3 pyramidal neuron responsivity was observed in the heterozygous mice compared to the wild type mice. These data suggest that decreasing hippocampal alpha7 receptor density results in a profound alteration in hippocampal circuit function.

Cloning and expression of streptomycin inactivating enzymes APH(6)-Ia and APH(6)-Id.

Discovered in the 1940s by Selman Waksman, the aminoglycoside antibiotic streptomycin is clinically important in the treatment of tuberculosis worldwide. However, strains of Mycobacterium tuberculosis and other pathogenic bacteria have become resistant to streptomycin. One mechanism by which this can occur is through the action of phosphotransferases that attach a phosphate group to position 6 of the streptidine ring of streptomycin, thereby inactivating it. Two such phosphotransferases are APH(6)-Ia from producer strain Streptomyces griseus, and APH(6)-Id found in animal, plant and human pathogenic isolates. Here, we report the subcloning and expression in Escherichia coli of soluble recombinant APH(6)-Ia and Id enzymes. Sequencing of aph(6)-Ia revealed a one-nucleotide disagreement with the published sequence, such that the amino acid at position 262 is an alanine instead of a serine. The sequence of aph(6)-Id is identical to that of the gene found in transposon Tn5393 of plant pathogen Erwinia amylovora. The successful expression of soluble forms of these enzymes now paves the way for experiments to study their structure and function by using site-directed mutagenesis.

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.

Atelinae phylogenetic relationships: the trichotomy revived?

This research examines phylogenetic relationships between members of the Atelinae subfamily (Alouatta, Ateles, Brachyteles, and Lagothrix), based on analysis of three genetic regions. Two loci, cytochrome c oxidase subunit II (COII) and the hypervariable I portion of the control region, are part of the mitochondrial genome. The other is a single-copy nuclear gene, Aldolase A Intron V. Analysis of these genetic regions provides support for tribe Alouattini containing the Alouatta species, while tribe Atelini contains the other three genera. However, these three genetic regions produce conflicting results for relationships among tribe Atelini members. Previous genetic studies supported grouping Brachyteles with Lagothrix, leaving Ateles in a separate subclade. The present data sets vary based on the genetic region analyzed and method of analysis suggesting all possible cladistic relationships. These results are more consistent with investigations of morphology and behavior among these primates. The primary cause of discrepancy between this study and previous genetic studies is postulated to reside in increased sampling in the present study of genetic variation among members of the Atelinae, specifically Ateles. The present study utilized samples of Ateles from all postulated species for this genetically variable primate, while previous studies used only one or two species of Ateles. This paper demonstrates that shifting relationships are produced when different species of Ateles are used to reconstruct phylogenies. This research concludes that a trichotomy should still be supported between members of tribe Atelini until further analyses, which include additional Atelinae haplotypes are conducted.

Alpha7-nicotinic receptor expression and the anatomical organization of hippocampal interneurons.

C3H and DBA/2 mice differ in their hippocampal inhibitory function, as measured by the inhibitory gating of pyramidal neuron response to repeated auditory stimulation. This functional difference appears to be related to differences in expression of the alpha7 nicotinic cholinergic receptor, which may be generally expressed by interneurons. This study examines the relationship between genetic variation in alpha7 receptor subunit expression and GABAergic interneuron distribution in various regions and layers of the hippocampus in the two mouse strains. Subpopulations of hippocampal interneurons in both mouse strains were found to bind [(125)I]alpha-bungarotoxin. However, the distribution of the [(125)I]alpha-bungarotoxin-positive hippocampal interneurons was significantly different between C3H and DBA/2 mice. In region CA1, and to a lesser extent in region CA3, DBA/2 mice had increased numbers of [(125)I]alpha-bungarotoxin-positive neurons in stratum lacunosum-moleculare and decreased numbers in stratum oriens. Similar differences in GABAergic neuron distribution were observed in region CA1 in the two strains. C3H/DBA/2 F1 animals were backcrossed to the C3H parental strain for six generations, with selection for either the DBA/2 or C3H allelic variant of the alpha7 receptor gene. The distribution of [(125)I]alpha-bungarotoxin labeling closely resembled the DBA/2 parental phenotype in animals retaining the DBA/2 allele of the alpha7 gene. These data suggest that the alpha7 receptor gene locus may influence the anatomical organization of at least a subset of hippocampal interneurons by an as yet unidentified mechanism. This difference in interneuron anatomy may also contribute to functional differences in inhibitory sensory gating between the two strains.

An aid to the early detection and management of diabetic nephropathy: assessment of a new point of care microalbuminuria system in the diabetic clinic.

AIMS: To compare the performance of the DCA2000 microalbuminuria system for albumin and creatinine concentrations and the albumin:creatinine ratio (ACR) with laboratory measurements in the hospital diabetes clinic and to assess the ease of use and applicability by standard clinic personnel. METHODS: Urine albumin and creatinine concentration and ACR were measured in 154 diabetic patient samples and in 77 normal subjects. Both albumin assays are based on immunoturbidimetry. The DCA2000 system utilizes reagent cartridges processed automatically. RESULTS: Control material within-run precision (coefficient of variation (CV)) for albumin and creatinine ranged up to 7.1% and 3.3% respectively. Between-run CVs ranged from 2.1% to 4.3%. Method comparisons yielded correlation coefficients > 0.99 for albumin, creatinine and ACR, only a small negative bias of 3.2 mg/l for albumin and 0.10 mg/mmol for ACR, no concentration-related bias for ACR and no between-method difference for either albumin (P = 0.195) or ACR (P = 0.341). At a laboratory albumin concentration cut-off of 20 mg/l the sensitivity, specificity, negative and positive predictive values were 92.4% 100% 92.7% and 100%. Normal reference range mean albumin, creatinine and ACR values for the DCA2000 and the laboratory were 7.7 mg/l vs. 9.0 mg/l 13.0 mmol/l vs. 12.6 mmol/l and 0.66 mg/mmol vs. 0.81 mg/mmol respectively. Clinic personnel found that the DCA2000 system was easy to use suited the clinic environment and generated confidence in the results. CONCLUSIONS: This point of care system safely substitutes laboratory-based measurements. Ease of use and low cost make it suitable for screening and monitoring diabetes treatment. It facilitates the use of random urines, and may obviate the need for timed samples. This approach has a clear place in the battle to reduce the diabetic vascular disease burden.

Chronic corticosterone treatment alters sensory gating in C3H mice.

Two methods of evaluating inhibitory sensory processing are prepulse inhibition of acoustic startle (PPI) and gating of auditory evoked potentials. Studies using both methods suggest nicotinic acetylcholinergic receptor modulation of gating, specifically the alpha-bungarotoxin (alpha-BTX) binding site (alpha7 receptor subtype). However, recent assessment of alpha7 null mutant mice failed to demonstrate any effect of the loss of this receptor in either gating paradigm. An alternate approach to assessing the effects of the alpha7 receptor is to reduce its numbers in mature inbred mice, thus, avoiding the twin problems of background and developmental compensation inherent in null mutant mouse studies. Numerous studies have shown that chronic corticosterone (CCS) treatment selectively reduces alpha-BTX binding sites. C3H mice were adrenalectomized and implanted with corticosterone or cholesterol (control) pellets. After 8 days, they were tested in one of the gating paradigms. PPI and auditory gating were significantly diminished in corticosterone-treated mice concomitant with a reduction in alpha-BTX binding in several brain regions. Cholesterol-treated mice had no change in either paradigm. Nicotine treatment (1 mg/kg) produced significant improvement in both paradigms in corticosterone-treated mice. These data agree with previous pharmacological studies suggesting modulation of gating occurs through a nicotinic receptor.

Potential regulation of nicotine and ethanol actions by alpha4-containing nicotinic receptors.

A restriction fragment length polymorphism (RFLP) associated with a major nicotinic receptor subunit (i.e., alpha4) has been identified in two mouse lines that were selectively bred for differences in sensitivity to ethanol. These mice, referred to as Long-Sleep (LS) and Short-Sleep (SS) mice, also differ in sensitivity to several effects of nicotine. The potential role of the alpha4 RFLP in regulating several responses to nicotine and ethanol was evaluated by using the LSxSS-derived recombinant inbred (RI) strains. Those RI strains that carried the LS-like alpha4 RFLP were more sensitive to the depressant effects of nicotine on Y-maze crossing and rearing activities and ethanol-induced increases in Y-maze crossing activity than were those RI strains that carry the SS-like alpha4 RFLP. The LS-like RI strains were also more sensitive to nicotine-induced hypothermia. The RFLP was not associated with strain differences in ethanol-induced body temperature or sleep time. The potential role of the RFLP in regulating ethanol and nicotine consumption was evaluated in heterogeneous stock (HS) mice. An association was found between the alpha4 RFLP and variation in ethanol consumption, but not in nicotine consumption, as measured in a four-bottle choice test. Recent studies of ethanol and tobacco abuse by human beings suggest that common genes may influence these two forms of substance abuse. The results of the studies reported here suggest that the alpha4 nicotinic receptor gene should be evaluated for its potential role in regulating ethanol and tobacco abuse in human beings.

Long sleep and short sleep mice differ in nicotine-stimulated 86Rb+ efflux and alpha4 nicotinic receptor subunit cDNA sequence.

In a recent study, we reported that a restriction fragment length polymorphism associated with the alpha4 nicotinic receptor gene (Chrna4) may play a role in regulating differential sensitivity of LS and SS mouse lines to the seizure-inducing effects of nicotine. Since the alpha4 subunit (CHRNA4) is often found as a heteromer with the beta2 subunit (CHRNB2), alpha4 and beta2 cDNAs from the LS and SS mice were cloned and sequenced. A polymorphism in the coding portion of the alpha4 gene was found (1587A to G) which should result in a threonine/alanine substitution at position 529 (T529A). The LS and SS beta2 nicotinic receptor subunit cDNAs were identical. The potential consequences of the alpha4 polymorphism were evaluated using an ion (86Rb+) flux assay that likely measures the function of alpha4beta2-type receptors. LS-SS differences in maximal nicotine-stimulated ion flux were seen when bovine serum albumin (BSA) was not included but this difference was not seen when BSA was included in the perfusion buffer. Current evidence suggests that BSA may alter the ratio of nicotinic receptors that are in the ground state and desensitized forms. Thus, it may be that the Chrna4 T529A substitution leads to a difference in the ratio of the two receptor forms which then promotes differences in receptor function, as well as differential behavioural sensitivity to nicotine.

Nuclear DNA variation in spider monkeys (Ateles).

Phylogenetic relationships based on DNA sequence variation for the aldolase A intron V nuclear genomic region were evaluated and compared to phylogenies based on mitochondrial DNA sequence variation among spider monkeys (Ateles). Samples of Ateles ranging from Central America throughout the Amazon Basin were sequenced to determine phylogenetic relationships among geographically widely distributed populations. Analysis of nuclear DNA sequences using parsimony, maximum-likelihood, and genetic distance analyses produced similar phylogenies. Four previously proposed monophyletic species of spider monkeys were: (1) Ateles paniscus, composed of haplotypes from the northeastern Amazon Basin; (2) A. belzebuth, found in the western and southern Amazon Basin; (3) A. hybridus, located primarily along the Magdalena River valley of Colombia; and (4) A. geoffroyi, including all haplotypes found in the Choco region of South America and throughout Central America. The nuclear phylograms were analyzed based on associated bootstrap support and confidence probabilities. Support from the nuclear DNA genome was less robust than support from the mitochondrial DNA data, most likely due to a level of sequence variation, which was 90% less than that of the mitochondrial DNA genome. Nuclear DNA congruencies with mitochondrial DNA-based phylogenies, as supported by the incongruence length difference and winning sites tests, provide further support for the suggested revisions in Ateles taxonomy that are contradictory to long-held taxonomies based on pelage variation.

Nicotine enhances latent inhibition and ameliorates ethanol-induced deficits in latent inhibition.

Alcohol and nicotine are drugs of abuse that are used frequently together. One possible explanation for this co-administration is that nicotine prevents or lessens alcohol-associated impairments. The present study examined the dose-dependent effects of acute administration of nicotine, alcohol, or alcohol plus nicotine on latent inhibition as measured by lick suppression in C57BL/6 mice. Alterations in a lick suppression ratio were measured by assessing the effects of 10 pre-exposures to an auditory conditioned stimulus (CS) on formation of subsequent CS-shock unconditioned stimulus (US) associations. Mice pre-exposed to the CS were expected to develop a weaker CS-US association. Nicotine administered prior to pre-exposure to the CS produced increased suppression ratios, ethanol given prior to pre-exposure to the CS decreased suppression ratios, and nicotine reversed the effects of ethanol when the two drugs were co-administered. These opposing actions of nicotine and ethanol may have relevance to the high incidence of smoking and drinking in humans.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

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.

A method for fine mapping quantitative trait loci in outbred animal stocks.

High-resolution mapping of quantitative trait loci (QTL) in animals has proved to be difficult because the large effect sizes detected in crosses between inbred strains are often caused by numerous linked QTLs, each of small effect. In a study of fearfulness in mice, we have shown it is possible to fine map small-effect QTLs in a genetically heterogeneous stock (HS). This strategy is a powerful general method of fine mapping QTLs, provided QTLs detected in crosses between inbred strains that formed the HS can be reliably detected in the HS. We show here that single-marker association analysis identifies only two of five QTLs expected to be segregating in the HS and apparently limits the strategy's usefulness for fine mapping. We solve this problem with a multipoint analysis that assigns the probability that an allele descends from each progenitor in the HS. The analysis does not use pedigrees but instead requires information about the HS founder haplotypes. With this method we mapped all three previously undetected loci [chromosome (Chr.) 1 logP 4.9, Chr. 10 logP 6.0, Chr. 15 logP 4.0]. We show that the reason for the failure of single-marker association to detect QTLs is its inability to distinguish opposing phenotypic effects when they occur on the same marker allele. We have developed a robust method of fine mapping QTLs in genetically heterogeneous animals and suggest it is now cost effective to undertake genomewide high-resolution analysis of complex traits in parallel on the same set of mice.

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.

Mice lacking PKC gamma exhibit decreased anxiety.

To investigate the contribution of the PKC gamma isoform of protein kinase C (PKC) in neurochemical pathways regulating anxiety, mice lacking the gene encoding PKC gamma were tested with heterozygote and wild-type littermates in three approach-avoidance tests of anxiety. Null mutant mice consistently displayed a decrease in baseline anxiety-related behaviors in the elevated plus-maze, the black/white box, and the mirrored chamber. In the elevated plus-maze, mutant mice entered the open arms significantly more often and spent more time in the open arms of the maze. In the black/white box, transitions between the compartments were greatest in the null mutant mice, and in the mirrored chamber, mutant mice were markedly less anxious with significantly decreased latencies to enter and more time spent in the chamber. Indices of locomotor activity in the mazes and tests of activity in home cages indicated that the reduced anxiety observed in the mutant mice was not due to baseline locomotor activity differences among the genotypes. These results suggest that PKC gamma be considered as one factor in the etiology of anxiety, perhaps via its post-synaptic regulation of GABAA and 5-HT2 receptors, two receptors implicated in the neurobiology of anxiety.

Genetic and pharmacological strategies identify a behavioral function of neuronal nicotinic receptors.

The studies outlined here used pharmacological and genetic approaches to attempt to identify the nicotinic receptors that modulate nicotine-induced seizures. Full-blown clonic-tonic seizures were induced by intracerebroventricular (i.c.v.) injection of nicotine, the alpha4beta2 selective agonist ABT-418 and the alpha7-selective GTS-21. Cytisine, which is a partial agonist at alpha4beta2-type receptors, produced partial seizures. DHbetaE and MLA did not block nicotine-induced seizures. Instead, both antagonists caused seizures. Restriction fragment length polymorphisms (RFLPs) for the alpha7 receptor were identified in two inbred strains (C3H and DBA) that differ in sensitivity to nicotine-induced seizures. F2 mice derived from a C3H x DBA cross that were homozygous for the C3H variant of the alpha7 RFLP were more sensitive to nicotine-induced seizures than were F2 mice that were homozygous for the DBA RFLP. In a study that used RI strains derived from two selectively bred mouse lines (LS and SS), an association between sensitivity to nicotine-induced seizures and an RFLP associated with the alpha4 gene was found. These data support the assertion that both alpha4 and alpha7 receptor types are involved in modulating convulsions produced by nicotine.

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.