WWW.JNEUROSCI.ORG
-
The Journal of Neuroscience
QUICK SEARCH:   [advanced]


     
-


HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (241)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orr-Urtreger, A.
Right arrow Articles by Beaudet, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orr-Urtreger, A.
Right arrow Articles by Beaudet, A. L.

 Previous Article  |  Next Article 

Volume 17, Number 23, Issue of December 1, 1997

Mice Deficient in the alpha 7 Neuronal Nicotinic Acetylcholine Receptor Lack alpha -Bungarotoxin Binding Sites and Hippocampal Fast Nicotinic Currents

Avi Orr-Urtreger1, 4, Finn M. Göldner2, Mayuko Saeki2, Isabel Lorenzo1, 4, Leah Goldberg1, 4, Mariella De Biasi3, John A. Dani2, James W. Patrick2, and Arthur L. Beaudet1, 4

1 Department of Molecular and Human Genetics, 2 Division of Neuroscience, and 3 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, and 4 The Howard Hughes Medical Institute, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The alpha 7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha -bungarotoxin (alpha -BGT). A null mutation for the alpha 7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha -BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha -BGT binding sites are equivalent to the alpha 7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha 7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.

Key words: acetylcholine receptor; alpha -bungarotoxin; gene targeting; hippocampus; mouse; nicotine; alpha 7 subunit

INTRODUCTION

Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of ligand-gated ion channels that include muscle and neuronal nAChRs and receptors for GABAA, glycine, and serotonin. Eleven genes have been identified that encode neuronal nAChR subunits: eight alpha  subunits (alpha 2-alpha 9) and three beta  subunits (beta 2-beta 4) (Sargent, 1993; McGehee and Role, 1995). Transcripts for alpha 8 have been found in avians but not in mammals (Schoepfer et al., 1990), and alpha 9-containing nAChRs are expressed in auditory hair cells (Elgoyhen et al., 1994). In the vertebrate brain, two categories of nicotinic receptors are distinguishable based on high-affinity binding of the agonist nicotine or high-affinity binding of the antagonist alpha -bungarotoxin (alpha -BGT). The receptors from these two categories have distinct distributions (Wonnacott, 1986; Whiting and Lindstrom, 1988).

Biochemical and electrophysiological investigations have established that both neuronal and skeletal muscle nAChRs are heteromultimers consisting of five subunits each with four membrane-spanning segments (Sargent, 1993; McGehee and Role, 1995). Expression studies in Xenopus oocytes demonstrated that functional neuronal nAChRs are comprised of two alpha  subunits and three beta  subunits (Cooper et al., 1991; Bertrand and Changeux, 1995). The alpha 7 subunit is an exception because it apparently cannot coassemble with other subunits when expressed in Xenopus oocytes. Thus, like alpha 8 and alpha 9, alpha 7 forms a homo-oligomer receptor/channel that is inhibited with high affinity by alpha -BGT (Couturier et al., 1990; Séguéla et al., 1993). Because alpha 7 is the only subunit of these three that is widely expressed in mammalian brain, it has been suspected that alpha 7 contributes to the high-affinity alpha -BGT binding site, the structure and functional significance of which have been a long-standing source of controversy (Sargent, 1993).

Initially, it was thought that the alpha -BGT sites did not form functional ion channels, but more recent studies have demonstrated high-affinity inhibition by alpha -BGT and methyllycaconitine (MLA) of a rapidly desensitizing nicotinic current in hippocampal neurons (Albuquerque and Alkondon, 1991; Zorumski et al., 1992; Alkondon and Albuquerque, 1993; Gray et al., 1996) and in neurons of the peripheral nervous system (Zhang et al., 1994). Previous studies based on protein purification indicated that alpha -BGT sites were hetero-oligomers, but the copurified components may not have been integral subunits (Sargent, 1993). In chick brain and retina, alpha -BGT sites were found to be heterogeneous, containing alpha 7, alpha 8, or both, possibly in combination with another unknown component (Schoepfer et al., 1990; Gotti et al., 1995).

Despite morphological, biochemical, and electrophysiological analysis, the functions of neuronal alpha -BGT binding sites and nAChRs in the mammalian brain remain largely unknown. Various neuronal nAChRs are involved in nicotine addiction (Dani and Heinemann, 1996), and there is evidence that nicotine can improve attention, rapid information processing, and working memory (Levin, 1992; Ohno et al., 1993; Picciotto et al., 1995; McGehee and Role, 1996). Cholinergic mechanisms may be involved in neurobehavioral disorders including seizures and schizophrenia (Steinlein et al., 1995; Freedman et al., 1997). A locus for juvenile myoclonic epilepsy is mapped near the alpha 7 gene in humans (Elmslie et al., 1997). Some functions attributed to the alpha -BGT sites and/or the alpha 7 subunit include synapse formation (Fuchs, 1989; Pugh and Berg, 1994; Broide et al., 1995), mediation of nicotine-induced seizures (Miner and Collins, 1989; Stitzel et al., 1997), and presynaptic modulation of neurotransmitter release (McGehee et al., 1995; Gray et al., 1996; Zhang et al., 1996).

We prepared mice with a homozygous null mutation for the alpha 7 subunit and found that the mice were viable and had no gross abnormalities in brain morphology, but the high-affinity alpha -BGT binding sites were not present. Furthermore, alpha 7-deficient hippocampal neurons lack fast, rapidly desensitizing nicotinic currents, indicating that those currents in the hippocampus are derived from alpha 7-containing nAChRs that are also likely to be the major high-affinity alpha -BGT binding sites in mouse brain.

MATERIALS AND METHODS

Acralpha 7 gene targeting in embryonic stem cells. A rat cDNA clone for the alpha 7 subunit (Séguéla et al., 1993) was used to screen a mouse genomic DNA library prepared from the 129/SvJ strain (cat. #946305, Stratagene, La Jolla, CA). Detailed restriction maps were prepared for genomic clones, and the intron/exon boundaries of exons 5-10 and the 3'-untranslated region in exon 10 were sequenced (Fig. 1a); sequences agreed with that published for the mouse alpha 7 cDNA (Orr-Urtreger et al., 1995). Sequencing was performed using an Applied Biosystems (ABI) model 373-automated DNA sequencer and dye terminator protocols as provided by the manufacturer (ABI, Foster City, CA).
Fig. 1. Gene targeting of the neuronal nAChR alpha 7 subunit. a, Partial genomic structure of the murine alpha 7 subunit gene including exons 5-10 is shown. The homologous recombination event generated a 7 kb genomic deletion that removes exons 8-10. Restriction enzyme sites are as follows: E, EcoRI; S, SacI; Sp, SpeI; B, BamHI. The diagnostic probes include a flanking probe to genotype ES cells and animals (external probe 1, Ext-1) and two internal probes (Int-1 and Int-2) to confirm the deletion. The targeting vector was used to obtain a replacement mutation and contains a neomycin resistance gene (neor) as a positive selectable marker and the herpes simplex thymidine kinase gene (HSV-tk) as a negative selectable marker. The sites of predicted homologous recombination are shown. The expected wild-type and mutant restriction fragments, after SacI enzyme digest and hybridization with Ext-1 probe, are shown below the targeting vector. b, Southern blot analysis identifies the alpha 7 homozygous null (-/-), heterozygous (+/-), and littermate control (+/+) animals using each of the three probes as indicated. The small arrow indicates the mutant band with the Ext-1 probe. Constant fragments of 2.0 and 0.3 kb are seen with the Ext-1 probe. c, Northern blot analysis of alpha 7 gene expression in brains of (+/+), (+/-), and (-/-) animals, using the Acralpha 7 cDNA (Acra7) and control (Gapdh) probes. d, Western blot analysis of brains from +/+ and -/- animals. As a positive control, 10, 20, 30, and 40 ng of recombinant extracellular domain of the rat alpha 7 protein (Chen and Patrick, 1997) were used.
[View Larger Version of this Image (36K GIF file)]

The deletion mutation was introduced into the ABI 2.1 embryonic stem (ES) cell line (Soriano et al., 1991) and transmitted to the germline as described previously (Bullard et al., 1996). Chimeric mice were bred with C57BL/6J mice, and the mice used in these studies were maintained on a mixed 129/SvEv and C57BL/6J background. The mutation is being back-crossed on the C57BL/6J background for future work.

Southern, Northern, and Western analyses. Southern blot hybridization was performed according to standard methods (Sambrook et al., 1989) using a hybridization solution of 0.125 M NaPO4, pH 7.0, 0.25 M NaCl, 1 mM EDTA, 10% polyethylene glycol (PEG-8000), 7% SDS, and 1% bovine serum albumin (BSA) at 65°C overnight followed by washing to a final stringency of 0.2× SSC/0.1% SDS at 65°C and autoradiography at -80°C.

Total RNA was isolated from brain after homogenization in Ultraspec II (Tel Test, Houston, TX). Total RNA was resolved on a 1.2% agarose gel in 10 mM NaPO4 buffer, pH 6.8, after glyoxal/dimethylsulfoxide denaturation according to standard methods (Sambrook et al., 1989). RNA was visualized by ethidium bromide staining, transferred to Hybond N+ membrane, and hybridized with full-length (mouse) Acralpha 7 cDNA as probe. Hybridization and washing conditions were identical to those used for Southern hybridization.

For immunoblotting, whole brain from three homozygous alpha 7 null (-/-) mice and three littermate control (+/+) mice was homogenized in 10 ml of ice-cold buffer containing 10 mM HEPES, pH 7.4, 5 mM EDTA, 5 mM EGTA, and 1 mM phenylmethylsulfonylfluoride (PMSF). Total membranes were pelleted by centrifugation at 100,000 × g for 60 min at 4°C. Membranes were resuspended in 10 ml of solubilization buffer containing 10 mM HEPES, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1% Triton X-100, and 1 mM PMSF and shaken for 2 hr at 4°C. The detergent-solubilized membrane protein supernatant was recovered after centrifugation at 100,000 × g for 60 min. Protein concentration was determined by the BCA method (Pierce, Rockford, IL). Solubilized membrane proteins (36 mg) were incubated overnight with 25 µl of alpha -cobratoxin matrix (0.5 mg toxin/ml matrix, Sigma, St. Louis, MO), which was preequilibrated with the solubilization buffer. The matrix was recovered by brief centrifugation in a benchtop microcentrifuge and washed four times with 1 ml of solubilization buffer. Bound receptors were eluted with 30 µl of SDS/sample buffer and separated by SDS-PAGE followed by transfer to a nitrocellulose membrane. The membrane was blocked for 30 min with 5% dry milk and 0.1% Tween 20 in PBS. A polyclonal sheep antibody (Chen and Patrick, 1997) against a bacterial expressed extracellular domain of the rat alpha 7 protein was diluted 1:1000 in the above blocking solution and incubated with the membrane overnight at 4°C. The membrane was washed and probed with a rabbit anti-sheep IgG antibody coupled to peroxidase (Cappel, Durham, NC) at a dilution of 1:10,000. After we washed the membranes, we detected peroxidase activity using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).

Nicotine and alpha -BGT autoradiography. For nicotine autoradiography, coronal sections (10 µm) of fresh frozen mouse brain were prepared and dried onto slides. [3H]nicotine (75 Ci/mmol, DuPont NEN, Boston, MA) was diluted in 50 mM Tris, pH 7.4, and 1% BSA to a final concentration of 2 nM. Slides were incubated with the solution for 1 hr at room temperature. Nonspecific binding was assessed by incubating sections with an excess (10 µM) of unlabeled ligand nicotine. Slides were washed twice in ice-cold 50 mM Tris, pH 7.4, for 2 min each, dried, and exposed to tritium-sensitive film (3H Hyperfilm, Amersham, Arlington Heights, IL) for 12 weeks.

For alpha -BGT autoradiography, unfixed, frozen brain was sectioned at 20 µm, thaw-mounted onto slides, and dried. Sections were preincubated for 30 min at room temperature in 1% BSA and 50 mM Tris, pH 7.4, either with or without 10 µM unlabeled alpha -BGT as a competitor. [125I]alpha -BGT (initial specific activity of 4.8 × 108 cpm/nmol and used within 1 half-life; Amersham) was diluted in the above blocking solution to a final concentration of 5 nM and added to the sections for 1 hr at room temperature. Sections were washed four times for 5 min in ice-cold 50 mM Tris, pH 7.4, after which they were dried and exposed to Kodak BioMax MR film for 1-3 d.

Histological analysis. Brain tissue for Nissl stain, immunohistochemistry, and acetylcholinesterase histochemistry was prepared by perfusion with PBS/4% paraformaldehyde in PBS, pH 7.4, and post-fixed overnight at 4°C in the same fixative. Tissue was equilibrated in 30% sucrose, frozen in prechilled isopentane, and sectioned at 30-50 µm.

For Nissl stains, cleared and rehydrated sections were incubated with 0.5% cresyl violet acetate, washed, rapidly dehydrated, cleared, and mounted on coverslips. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was performed with antibody against GFAP (clone G-A-C, mouse IgG1, Boehringer Mannheim, Indianapolis, IN); free-floating sections were stained according to a standard ABC-protocol (Vector Laboratories, Burlingame, CA). Staining for acetylcholine esterase was performed according to a published protocol (Geneser-Jensen and Blackstad, 1971). A modified Timm stain protocol was used to demonstrate mossy fibers in paraffin-embedded, 8-µm-thick sections of the hippocampus (Sloviter, 1982).

Labeling for cytochrome oxidase was performed on mounted sections using PBS containing 4% sucrose, 25 µM cytochrome c (type III, Sigma), and 1 mM diaminobenzidine tetrahydrochloride at 37°C until staining of the desired intensity could be detected, after which the reaction was stopped by washing in PBS.

Electrophysiology. Hippocampal neurons were obtained from newborn mice (12-36 hr postnatal). The brain was removed immediately and kept throughout the dissection in a cold solution containing (in mM): 137 NaCl, 5.3 KCl, 0.2 Na2HPO4, 0.2 KH2PO4, and 10 HEPES, pH 7.4. Hippocampal tissue from both hemispheres was gently removed and cut into small pieces. The tissue was digested for 40 min at 37°C in a dissecting solution containing 20 U/ml papain (Worthington, Freehold, NJ). After digestion, the tissue was triturated with fire-polished Pasteur pipettes of decreasing diameter. Hippocampal cells were plated on collagen/poly-D-lysine-coated coverslips (Fisher, Pittsburgh, PA). A separate cell culture was prepared for each mouse in litters born to heterozygote parents, with the expectation that +/+, +/-, and -/- pups would occur in a ratio of 1:2:1. The genotype of each mouse was determined after electrophysiological studies were completed and interpreted. Cells were kept in minimum essential medium (MEM) containing 5% fetal bovine serum (HyClone, Logan, UT), 1 µl/ml Serum Extender (Collaborative Research, Bedford, MA), 0.5 µM tetrodotoxin (Calbiochem, Pasadena, CA), 20 mM glucose, and 2.5 mM MgCl2. Hippocampal neurons were studied from day 15 to day 25 in culture because nicotinic currents are larger and more commonly expressed after day 10 in culture (Alkondon and Albuquerque, 1993; Gray et al., 1996).

Whole-cell currents were elicited with 500 µM nicotine and measured with standard patch-clamp techniques (Zarei and Dani, 1995). The external bath solution contained (in mM): 150 NaCl, 2.5 KCl, 5 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH 7.4, 0.5 µM tetrodotoxin, and 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The pipette solution contained (in mM): 150 CsCH3SO3, 5 NaCl, 0.2 EGTA, 2 Na2ATP, 2 MgATP, 0.3 Na3GTP, and 10 HEPES, pH 7.3. The holding potential was -60 mV. Currents were amplified and filtered on line (1 kHz) using an Axopatch 1D voltage clamp with a 4-pole Bessel filter and were digitally sampled to exceed the Nyquist criterion. No series resistance compensation or leak subtraction was performed. Drugs were applied through flow pipes mounted on a motorized manipulator (Newport, Fountain Valley, CA) and connected to computer-driven valves (General Valve Corporation, Fairfield, NJ). Nicotine and MLA were applied for 600 msec and 90 sec, respectively. Solution changes were complete in ~30 msec (Gray et al., 1996). All experiments were conducted at room temperature.

RESULTS

Preparation of a null mutation for the alpha 7 subunit

Mice deficient in the alpha 7 subunit were generated by introducing a 7 kb deletion into ES cells followed by transmission to the germline. The mutation deletes the last three exons (8-10) of the alpha 7 locus (Acralpha 7); these exons encode the second transmembrane domain (MII), forming the putative ion channel and the third and fourth transmembrane domains and the cytoplasmic loop (Fig. 1a). Southern blot analysis using a flanking genomic probe detected a new 3.2 kb mutant fragment in the heterozygote (+/-) and homozygote (-/-) mice (Fig. 1b); adjacent smaller DNA fragments were unchanged. Analysis with genomic probes from the regions of the deleted exons confirmed the absence of these exons (Fig. 1b). The effect of the mutation on mRNA transcripts was examined using Northern blotting, and no detectable transcripts were found in homozygous mutant mice, indicating that any altered transcripts were unstable (Fig. 1c). Because the deletion leaves intact the coding sequences for the extracellular, ligand-binding, and toxin-binding domains of the alpha 7 subunit, a partially functional, truncated protein could occur; this possibility is unlikely, however, considering the results of Northern blotting. Immunoblots (Fig. 1d) of protein extracts prepared by alpha -BGT affinity chromatography from wild-type and mutant mice were probed with antibodies to the N-terminal portion of the alpha 7 subunit, demonstrating absence of toxin-binding alpha 7 protein in homozygous mice. The results of Northern analysis and immunoblotting confirm the generation of a null mutation for the alpha 7 subunit.

Normal growth, viability, and neuroanatomy in alpha 7 null mice

The alpha 7 null mice are viable, are present in the expected proportion in matings of heterozygote mice, grow to normal size, and show no obvious physical or neurological deficit. Homozygous male and female mice are fertile, although there may be some reduction in fertility.

The overall brain structure and organization in homozygous null mice appear normal as assessed by comparable Nissl-stained sections in +/+ and -/- mice (Fig. 2a). All major neuronal structures are intact, and there are no apparent abnormalities in cell density or layering of cortical structures. Because alpha 7 is expressed most abundantly in the hippocampus, a more detailed anatomical analysis was performed (Fig. 2b). Nissl staining revealed that the major classes of neurons (i.e., granule cells, pyramidal cells, and hilar interneurons) are present in their appropriate location and, therefore, do not appear to depend on the presence of alpha 7 for their formation or migration within the network. Histochemical staining for acetylcholinesterase (AChE) was performed to evaluate the hippocampus, and no abnormalities were found. Because a mutation in an alpha 7-like gene in C. elegans is associated with neurodegeneration (Treinin and Chalfie, 1995), we performed immunohistochemical staining for GFAP, a marker for astrocytes, to search for glial scars representing neuronal injury or cell death. No glial scars were found in the hippocampus of mutant mice, suggesting that deficiency of alpha 7 does not lead to abnormal neuronal degeneration in this region. The status of intrahippocampal connections, particularly the mossy fiber projections, was evaluated by Timm staining, and no differences were found between mutants and controls.

Fig. 2. Neuroanatomy and histochemistry of mutant mice. Panels with +/+ and -/- indicated are as follows: (a) coronal sections of brain with Nissl stain; (b) sagittal sections of hippocampus with Nissl stain (Nissl), AChE-acetylcholinesterase (AChE), glial fibrillary acidic protein (GFAP), and Timm stain (Timm) as indicated; and (c) sections through the barrels (arrows) in the primary somatosensory cortex stained with cytochrome oxidase.
[View Larger Version of this Image (73K GIF file)]

The primary somatosensory cortex of rodents is characterized by anatomically defined barrel structures that reflect projections from the whiskers. Because staining with alpha -BGT outlines barrels at the time of their appearance, it was suggested that alpha 7 may be important for barrel formation (Fuchs, 1989; Broide et al., 1995). Mutant animals were analyzed for the presence of barrels using histochemical staining for cytochrome oxidase (Fig. 2c). Sections through the somatosensory cortex of mutant animals show characteristic barrel-like structures that were indistinguishable from those in wild-type littermates, indicating that structures with the general appearance of barrels develop in the absence of alpha 7.

Null mice lack alpha -BGT binding sites

The distribution of high-affinity nicotine binding sites and alpha -BGT binding sites in the brain of mutant (-/-) and control (+/+) littermates was examined (Fig. 3). The pattern of distribution of binding sites for these two ligands is significantly different, with the greatest abundance of high-affinity nicotine binding sites in thalamic structures and the predominance of alpha -BGT binding sites in the hippocampus (Clarke et al., 1985). The use of [3H]nicotine at low nanomolar concentrations detects only high-affinity binding sites and does not detect low-affinity nicotine binding sites such as those present in hippocampus (Clarke et al., 1985; Picciotto et al., 1995). The pattern of high-affinity nicotine binding sites in mutant and control animals was indistinguishable, and there was no evidence for significant upregulation of nicotine binding sites in alpha 7-deficient mice (Fig. 3). The distribution of alpha -BGT binding sites in the alpha 7-deficient mice is of particular interest because of the possibility that alpha 7 represents the major or sole alpha -BGT binding protein in rodent brain. Brain sections of homozygous null mice and littermate controls were analyzed using autoradiography with [125I]alpha -BGT (Fig. 3). The characteristic distribution of alpha -BGT binding sites was found in normal mice with a prominent pattern in telencephalic structures such as the hippocampus, amygdala, and neocortex. In contrast, there was no significant alpha -BGT binding above background levels in the null mice, suggesting that alpha 7 is required for high-affinity alpha -BGT binding sites in the mouse brain.
Fig. 3. Nicotine and alpha -BGT binding in mutant mice. Autoradiography was performed with [3H]nicotine and [125I]alpha -BGT as described in Materials and Methods with +/+ mice shown on the left and -/- shown on the right. The panels marked cold alpha -BGT included excess nonradioactive alpha -BGT as competitor.
[View Larger Version of this Image (86K GIF file)]

Null mice lack hippocampal fast nicotinic currents

Electrophysiological evaluation of the mice was important, because rapidly desensitizing nicotinic currents in hippocampal neurons are attributed to expression of alpha 7 (Alkondon and Albuquerque, 1993; Gray et al., 1996). Neonatal littermates from matings of heterozygotes were examined, and genotypes were determined after collection and interpretation of the electrophysiological data. Hippocampal neurons were cultured from neonatal mice, and rapid application of 500 µM nicotine evoked a fast, desensitizing current that could be blocked by 5 nM MLA, a potent and selective antagonist of alpha 7-containing nAChRs (Alkondon and Albuquerque, 1993; Gray et al., 1996) (Fig. 4a). The block by MLA was reversible after 2 min of washout with bath solution. When neurons from alpha 7-deficient mice were exposed to 500 µM nicotine, no currents could be recorded; none of the 35 cells from the four null mice examined revealed any current (Fig. 4c). Neurons from both wild-type and heterozygous mice showed bimodal response distributions to nicotine; some cells responded by giving measurable currents, and other cells had no response. The lack of nicotine-induced currents in the alpha 7 null mice indicates that the predominant currents found in cultured hippocampal neurons from control mice are dependent on expression of the alpha 7 nAChR.
Fig. 4. Nicotine induces currents in control but not in alpha 7 null mice. a, Nicotine (0.5 mM) evoked a fast, desensitizing current in hippocampal neurons from control (+/+) mice. The currents were completely blocked by 5 nM MLA (90 sec application) and recovered to 89% of the original peak current after washout (recovery at 150 sec). The solid black lines indicate the duration of the nicotine applications. b, Nicotine (0.5 mM) failed to induce ionic currents in all of the hippocampal cells studied from alpha 7 null mice. c, The majority (72%) of hippocampal cells from wild-type (+/+) mice (3 animals) showed currents in response to nicotine application, and 28% did not show any response. Approximately half (54%) of hippocampal cells from alpha 7 heterozygous (+/-) mice (8 animals) responded to nicotine, whereas 46% did not show any current. No nicotine-induced current could be recorded in any of the cells from alpha 7 null (-/-) mice (4 animals, 35 cells). n, Number of cells recorded; I, current in pA.
[View Larger Version of this Image (22K GIF file)]

DISCUSSION

A mutation deleting numerous exons was introduced into the gene for the alpha 7 subunit, completely eliminating its potential for participation in an ion channel. Although this mutation might allow for the synthesis of a truncated protein with ligand-binding capacity, Northern blotting and immunoblotting studies demonstrate the absence of detectable mRNA or protein and ensure that this mutation produces a null allele. The function of the alpha 7 subunit is largely unknown, although many possible roles have been discussed (Sargent, 1993; McGehee and Role, 1995). Homozygous mutant mice demonstrate normal general appearance, growth, survival, gait, and anatomy. Histological evaluation of the nervous system did not reveal any developmental abnormalities. Thus, the phenotypic consequences of deficiency of the alpha 7 nAChR are not immediately obvious.

There is evidence that expression of the alpha 7 subunit may be correlated with differences in nicotine binding, nicotine-induced seizures, nicotine preference, and effect of nicotine on body temperature in various strains of mice (Miner and Collins, 1989; Stitzel et al., 1997), and it will be of interest to analyze the mutant mice for these traits. In addition, alpha 7-deficient mice might be expected to show resistance to alpha -conotoxin (Johnson et al., 1995). There is also a report suggesting that differences in response to auditory stimuli that are associated with schizophrenia show linkage to human chromosome 15 in a region near the alpha 7 locus (Freedman et al., 1997). There is evidence of increased smoking in schizophrenics, and alpha 7 has been proposed as having a role in the pathophysiology of schizophrenia (Freedman et al., 1994, 1997). It will be important to evaluate learning and behavior in the mutant mice, and the mutation is being back-crossed to C57BL/6J background, because nicotine-induced seizures, auditory gaiting, and learning are all known to be variable among inbred strains of mice (Miner and Collins, 1989; Stitzel et al., 1997). Behavioral studies seeking to identify schizophrenia-like behavior in mice (Dains et al., 1996; Kafka and Corbett, 1996) will also be of interest.

The hippocampus is a center for learning and memory and receives cholinergic innervation mainly from the medial septum and diagonal band (Woolf, 1991). Presynaptic terminals containing choline acetyltranferase (the enzyme that catalyzes the synthesis of acetylcholine) have been found to synapse directly onto pyramidal and granule cells and their dendrites (Alonso and Amaral, 1995). Nicotine and cytisine autoradiography and in situ hybridization using probes for various subunits indicate that nAChRs are expressed throughout the hippocampus and that alpha 7 and beta 2 are the most abundant subunits (Deneris et al., 1988; Wada et al., 1990; Dineley-Miller and Patrick, 1992; Perry et al., 1993; Séguéla et al., 1993). The hippocampus also is known to possess a high density of alpha -BGT binding sites. Our results with alpha 7 null mice indicate that the alpha -BGT sites are not detected when the alpha 7 gene is disrupted, but the high-affinity nicotine sites in the brain are not detectably different. Although the results do not eliminate the possibility of other low-affinity sites or of another very minor component of alpha -BGT sites, the major alpha -BGT sites that have been at the center of attention and controversy are absent in alpha 7 null mice. These results indicate that, unlike chick, in which multiple forms of alpha -BGT sites are seen based on the presence of either the alpha 7 or the alpha 8 subunit (Schoepfer et al., 1990; Gotti et al., 1995), the alpha -BGT sites require the alpha 7 subunit in mice.

In rat hippocampal slices and cultures, it was found that nicotinic agonists can enhance glutamate release by acting through presynaptic nAChRs (Gray et al., 1996). This enhanced release of glutamate and the rapidly desensitizing nicotinic currents were inhibited by alpha -BGT and MLA, indicating that those nAChRs contain the alpha 7 subunit. Although other types of nicotinic currents occasionally could be seen, the predominant current displayed fast activation and rapid desensitization (Alkondon and Albuquerque, 1993; Gray et al., 1996). Similarly for mice, we found nicotine-activated currents in the majority (72%) of hippocampal neurons, and in all cases those currents were rapid and inhibited by MLA. In cultures from four alpha 7 null mice, however, none of the 35 hippocampal neurons that were studied displayed nicotinic currents. These results suggest that the rapid nicotinic currents in hippocampal neurons are mediated by the alpha -BGT binding sites and that those sites require the alpha 7 subunit for their formation into a receptor/ion channel complex.

Whether homo-oligomer alpha 7 receptors exist in the brain is still open to question, but evidence is mounting that alpha 7 might form a receptor without requiring any of the other presently known nicotinic subunits. The alpha 7 null mice lack the alpha -BGT sites but have the high-affinity nicotine sites, and beta 2 null mice lack the nicotine sites but still have the alpha -BGT sites (Picciotto et al., 1995). These results indicate that beta 2 is not required for the alpha -BGT site and that alpha 7 is not required for the high-affinity nicotine site. Because alpha 7 and beta 2 are by far the predominant subunits in the hippocampus, it is unlikely that any other known subunit is abundant enough to be present in all of the alpha -BGT sites in the hippocampus. Thus, it is unlikely that all of the alpha -BGT sites contain a known subunit other than alpha 7. In addition, alpha -BGT sites from PC12 cells showed size and pharmacological similarities to homo-oligomeric chimeric alpha 7 receptors expressed in tsA201 cells (Rakhilin et al., 1996), further supporting the hypothesis that homo-oligomeric alpha 7 receptors may exist naturally as well as in heterologous expression systems.

Nicotine obtained from tobacco has complex psychopharmacological effects (Dani and Heinemann, 1996), but it is reasonable to hypothesize that nicotine acts, in part, on the hippocampus (Gray et al., 1996) to enhance learning and memory on various tasks (Levin, 1992; Ohno et al., 1993). The results of the present work suggest that the actions of nicotine on the hippocampus are likely to arise largely from activation, desensitization, or modification of alpha -BGT binding sites that contain the alpha 7 subunit.

Although it may be surprising that mice with complete deficiency of the alpha 7 subunit do not have gross or obvious abnormalities, these mice do demonstrate that the alpha 7 subunit is not essential for normal development or for superficially normal neurological function. These mice may prove to have subtle phenotypic abnormalities, and they will be valuable in defining the functional role of the alpha 7 subunit in vivo.

FOOTNOTES

Received July 31, 1997; revised Sept. 16, 1997; accepted Sept. 18, 1997.

  

This work was supported by National Institutes of Health Grants NS-21229, DA-09411, DA-04077, and TW-04861.

Correspondence should be addressed to Dr. Arthur L. Beaudet, Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Room T619, Houston, TX 77030.

Dr. Orr-Urtreger's present address: Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.


REFERENCES

  • Albuquerque EX, Alkondon M (1991) Initial characterization of the nicotinic acetylcholine receptors in rat hippocampal neurons. J Recept Res 11:1001-1021[Web of Science][Medline].
  • Alkondon M, Albuquerque EX (1993) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J Pharmacol Exp Ther 265:1455-1473[Abstract/Free Full Text].
  • Alonso JR, Amaral DG (1995) Cholinergic innervation of the primate hippocampal formation. I. Distribution of choline acetyltransferase immunoreactivity in the Macaca fascicularis and Macaca mulatta monkeys. J Comp Neurol 355:135-170[Web of Science][Medline].
  • Bertrand D, Changeux J-P (1995) Nicotinic receptor: an allosteric protein specialized for intercellular communication. Neuroscience 7:75-90.
  • Broide RS, O'Connor LT, Smith MA, Smith JAM, Leslie I (1995) Developmental expression of alpha 7 neuronal nicotinic receptor messenger RNA in rat sensory cortex and thalamus. Neuroscience 67:83-94[Web of Science][Medline].
  • Bullard DC, Kunkel EJ, Kubo H, Hicks MJ, Lorenzo I, Doyle NA, Doerschuk CM, Ley K, Beaudet AL (1996) Infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin double mutant mice. J Exp Med 183:2329-2336[Abstract/Free Full Text].
  • Chen D, Patrick JW (1997) The alpha-bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the alpha7 subunit. J Biol Chem 272:24024-24029[Abstract/Free Full Text].
  • Clarke PBS, Schwartz RD, Paul SM, Pert CB, Pert A (1985) Nicotinic binding in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine, and [125I]-alpha -bungarotoxin. J Neurosci 5:1307-1315[Abstract].
  • Cooper E, Couturier S, Ballivet M (1991) Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature 350:235-238[Medline].
  • Couturier S, Bertrand D, Matter J-M, Hernandez M-C, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M (1990) A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha -BTX. Neuron 5:847-856[Web of Science][Medline].
  • Dains K, Hitzemann B, Hitzemann R (1996) Genetics, neuroleptic response and the organization of cholinergic neurons in the mouse striatum. J Pharmacol Exp Ther 279:1430-1438[Abstract/Free Full Text].
  • Dani JA, Heinemann S (1996) Molecular and cellular aspects of nicotine abuse. Neuron 16:905-908[Web of Science][Medline].
  • Dineley-Miller K, Patrick J (1992) Gene transcripts for the nicotinic acetylcholine receptor subunit, beta 4, are distributed in multiple areas of the rat central nervous system. Mol Brain Res 16:339-344[Medline].
  • Elgoyhen AB, Johnson DS, Boulter J, Vetter DE, Heinemann S (1994) alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells. Cell 79:705-715[Web of Science][Medline].
  • Elmslie FV, Rees M, Williamson MP, Kerr M, Kjeldsen MJ, Pang KA, Sundqvist A, Friis ML, Chadwick D, Richens A, Covanis A, Santos M, Arzimanoglou A, Panayiotopoulos CP, Curtis D, Whitehouse WP, Gardiner RM (1997) Genetic mapping of a major susceptibility locus for juvenile myoclonic epilepsy on chromosome 15q. Hum Mol Genet 6:1329-1334[Abstract/Free Full Text].
  • Freedman R, Adler LE, Bickford P, Byerley W, Coon H, Cullum CM, Griffith JM, Harris JG, Leonard S, Miller C, Myles-Worsley M, Nagamoto HT, Rose G, Waldo M (1994) Schizophrenia, nicotinic receptors, and cigarette smoking. Harvard Rev Psychiatry 2:179-192.[Web of Science][Medline]
  • Freedman R, Coon H, Myles-Worsley M, Orr-Urtreger A, Olincy A, Davis A, Polymeropoulos M, Holik J, Hopkins J, Hoff M, Rosenthal J, Waldo MC, Reimherr F, Wender P, Yaw J, Young DA, Breese CR, Adams C, Patterson D, Adler LE, Kruglyak L, Leonard S, Byerley W (1997) Linkage of a neurophysiological deficit in schizophrenia to a chromosome 15 locus. Proc Natl Acad Sci USA 94:587-592[Abstract/Free Full Text].
  • Fuchs JL (1989) [125I]alpha -bungarotoxin binding marks primary sensory areas of developing rat neocortex. Brain Res 501:223-234[Web of Science][Medline].
  • Geneser-Jensen FA, Blackstad TW (1971) Distribution of acetylcholinesterase in the hippocampal region of the guinea pig. I. Entorhinal area, parasubiculum, and presubiculum. Z Zellforsch 114:460-481.[Web of Science][Medline]
  • Gotti C, Hanke W, Moretti M, Longhi R, Balestra B, Briscini L, Clementi F (1995) alpha -Bungarotoxin receptor subtypes. In: Effects of nicotine on biological systems, Pt II (Clarke PBS, Quik M, Adlkofer F, Thurau K, eds), pp 37-44. Boston: Birkhauser Verlag.
  • Gray R, Rajan AS, Radcliffe KA, Yakehiro M, Dani JA (1996) Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383:713-716[Medline].
  • Johnson DS, Martinez J, Elgoyhen AB, Heinemann SF, McIntosh JM (1995) alpha -Conotoxin Imi exhibits subtype-specific nicotinic acetylcholine receptor blockade: preferential inhibition of homomeric alpha 7 and alpha 9 receptors. Mol Pharmacol 48:194-199[Abstract].
  • Kafka SH, Corbett R (1996) Selective adenosine A2A receptor/dopamine D2 receptor interactions in animal models of schizophrenia. Eur J Pharmacol 295:147-154[Web of Science][Medline].
  • Levin DL (1992) Nicotinic systems and cognitive function. Psychopharmacology 108:417-431[Medline].
  • McGehee DS, Heath MJS, Gelber S, Devay P, Role LW (1995) Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors. Science 269:1692-1696[Abstract/Free Full Text].
  • McGehee DS, Role LW (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 57:521-546[Web of Science][Medline].
  • McGehee DS, Role LW (1996) Memories of nicotine. Nature 383:670-671[Medline].
  • Miner LL, Collins AC (1989) Strain comparison of nicotine-induced seizure sensitivity and nicotinic receptors. Pharmacol Biochem Behav 33:469-475[Web of Science][Medline].
  • Ohno M, Yamamoto T, Watanabe S (1993) Blockade of hippocampal nicotinic receptors impairs working memory but not reference memory in rats. Pharmacol Biochem Behav 45:89-93[Web of Science][Medline].
  • Orr-Urtreger A, Seldin MF, Baldini A, Beaudet AL (1995) Cloning and mapping of the mouse alpha 7-neuronal acetylcholine receptor. Genomics 26:399-402[Web of Science][Medline].
  • Picciotto MR, Zoll M, Lena C, Bessis A, Lallemand Y, LeNovere N, Vincent P, Pich EM, Brulet P, Changeux J-P (1995) Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain. Nature 374:65-67[Medline].
  • Pugh PC, Berg DK (1994) Neuronal acetylcholine receptors that bind alpha -bungarotoxin mediate neurite retraction in a calcium-dependent manner. J Neurosci 14:889-896[Abstract].
  • Rakhilin SV, Atluri P, Drisdel RC, Ko E, Rangwala F, Salman WN, Green WN (1996) alpha 7/5HT3 chimeric homomers: similarity to PC12 alpha -bungarotoxin receptors. Neuroscience 22:1521.
  • Sambrook J, Fritsch EF, Maniatis T (1989) In: Molecular cloning. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  • Sargent PB (1993) The diversity of neuronal nicotinic acetylcholine receptors. Annu Rev Neurosci 16:403-443[Web of Science][Medline].
  • Schoepfer R, Conroy WG, Whiting P, Gore M, Lindstrom J (1990) Brain alpha -bungarotoxin binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily. Neuron 5:35-48[Web of Science][Medline].
  • Séguéla P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW (1993) Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13:596-604[Abstract].
  • Sloviter RS (1982) A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain. Brain Res Bull 8:771-774[Web of Science][Medline].
  • Soriano P, Montgomery C, Geske R, Bradley A (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693-702[Web of Science][Medline].
  • Steinlein OK, Mulley JC, Propping P, Wallace RH, Phillips HA, Sutherland GR, Scheffer IE, Berkovic SF (1995) A missense mutation in the neuroinal nicotinic acetylcholine receptor alpha 4 subunit is associated with autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet 11:201-203[Web of Science][Medline].
  • Stitzel JA, Robinson SF, Marks MJ, Collins AC (1997) Differences in response to nicotine are determined by genetic factors. Adv Pharmacol Sci 279-284.
  • Treinin M, Chalfie M (1995) A mutated acetylcholine receptor subunit causes neuronal degeneration in C. elegans. Neuron 14:871-877[Web of Science][Medline].
  • Whiting PJ, Lindstrom JM (1988) Characterization of bovine and human neuronal nicotinic acetylcholine receptors using monoclonal antibodies. J Neurosci 8:3395-3404[Abstract].
  • Wonnacott S (1986) alpha -Bungarotoxin binds to low-affinity nicotine binding sites in rat brain. J Neurochem 47:1706-1712[Web of Science][Medline].
  • Woolf NJ (1991) Cholinergic systems in mammalian brain and spinal cord. Prog Neurobiol 37:475-524[Web of Science][Medline].
  • Zarei MM, Dani JA (1995) Structural basis for explaining open-channel blockage of the NMDA receptor. J Neurosci 15:1446-1454[Abstract].
  • Zhang Z-W, Coggan JS, Berg DK (1996) Synaptic currents generated by neuronal acetylcholine receptors sensitive to alpha -bungarotoxin. Neuron 17:1231-1240[Web of Science][Medline].
  • Zhang Z, Vijayaraghavan S, Berg D (1994) Neuronal acetylcholine receptors that bind alpha -bungarotoxin with high affinity function as ligand-gated ion channels. Neuron 12:167-177[Web of Science][Medline].
  • Zorumski CF, Thio LL, Isenberg KE, Clifford DB (1992) Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons. Mol Pharmacol 41:931-936[Abstract].
Copyright ©1997 Society for Neuroscience   0270-6474/1997/179165-07$05.00/0



This article has been cited by other articles:


Home page
 J. Immunol.Home page
E. Nizri, M. Irony-Tur-Sinai, O. Lory, A. Orr-Urtreger, E. Lavi, and T. Brenner
Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppression of Th1 and Th17 Responses
J. Immunol., November 15, 2009; 183(10): 6681 - 6688.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
K. Maouche, M. Polette, T. Jolly, K. Medjber, I. Cloez-Tayarani, J.-P. Changeux, H. Burlet, C. Terryn, C. Coraux, J.-M. Zahm, et al.
{alpha}7 Nicotinic Acetylcholine Receptor Regulates Airway Epithelium Differentiation by Controlling Basal Cell Proliferation
Am. J. Pathol., November 1, 2009; 175(5): 1868 - 1882.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. C. Barron, J. T. McLaughlin, J. A. See, V. L. Richards, and R. L. Rosenberg
An Allosteric Modulator of {alpha}7 Nicotinic Receptors, N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596), Causes Conformational Changes in the Extracellular Ligand Binding Domain Similar to Those Caused by Acetylcholine
Mol. Pharmacol., August 1, 2009; 76(2): 253 - 263.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
G. Dziewczapolski, C. M. Glogowski, E. Masliah, and S. F. Heinemann
Deletion of the {alpha}7 Nicotinic Acetylcholine Receptor Gene Improves Cognitive Deficits and Synaptic Pathology in a Mouse Model of Alzheimer's Disease
J. Neurosci., July 8, 2009; 29(27): 8805 - 8815.
[Abstract] [Full Text] [PDF]

Home page
 Cereb CortexHome page
G. Zolles, E. Wagner, A. Lampert, and B. Sutor
Functional Expression of Nicotinic Acetylcholine Receptors in Rat Neocortical Layer 5 Pyramidal Cells
Cereb Cortex, May 1, 2009; 19(5): 1079 - 1091.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
T. D. McClure-Begley, N. M. King, A. C. Collins, J. A. Stitzel, J. M. Wehner, and C. M. Butt
Acetylcholine-Stimulated [3H]GABA Release from Mouse Brain Synaptosomes is Modulated by {alpha}4{beta}2 and {alpha}4{alpha}5{beta}2 Nicotinic Receptor Subtypes
Mol. Pharmacol., April 1, 2009; 75(4): 918 - 926.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
D T Miller, Y Shen, L A Weiss, J Korn, I Anselm, C Bridgemohan, G F Cox, H Dickinson, J Gentile, D J Harris, et al.
Microdeletion/duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatric disorders
J. Med. Genet., April 1, 2009; 46(4): 242 - 248.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
E. X. Albuquerque, E. F. R. Pereira, M. Alkondon, and S. W. Rogers
Mammalian Nicotinic Acetylcholine Receptors: From Structure to Function
Physiol Rev, January 1, 2009; 89(1): 73 - 120.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
K. J. Jackson, B. R. Martin, J. P. Changeux, and M. I. Damaj
Differential Role of Nicotinic Acetylcholine Receptor Subunits in Physical and Affective Nicotine Withdrawal Signs
J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 302 - 312.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
C. Fischer, A. Fredriksson, and P. Eriksson
Coexposure of Neonatal Mice to a Flame Retardant PBDE 99 (2,2',4,4',5-Pentabromodiphenyl Ether) and Methyl Mercury Enhances Developmental Neurotoxic Defects
Toxicol. Sci., February 1, 2008; 101(2): 275 - 285.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. A. Davis, J. W. Kenney, and T. J. Gould
Hippocampal {alpha}4{beta}2 Nicotinic Acetylcholine Receptor Involvement in the Enhancing Effect of Acute Nicotine on Contextual Fear Conditioning
J. Neurosci., October 3, 2007; 27(40): 10870 - 10877.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Ma, J. Huang, J. Sun, G. Wang, C. Li, L. Xie, and R. Zhang
A Novel Extrapallial Fluid Protein Controls the Morphology of Nacre Lamellae in the Pearl Oyster, Pinctada fucata
J. Biol. Chem., August 10, 2007; 282(32): 23253 - 23263.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
J. Zhang and D. K. Berg
Reversible inhibition of GABAA receptors by {alpha}7-containing nicotinic receptors on the vertebrate postsynaptic neurons
J. Physiol., March 15, 2007; 579(3): 753 - 763.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
Z. Liu, R. A. Neff, and D. K. Berg
Sequential Interplay of Nicotinic and GABAergic Signaling Guides Neuronal Development
Science, December 8, 2006; 314(5805): 1610 - 1613.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
C. Le Magueresse, V. Safiulina, J.-P. Changeux, and E. Cherubini
Nicotinic modulation of network and synaptic transmission in the immature hippocampus investigated with genetically modified mice
J. Physiol., October 15, 2006; 576(2): 533 - 546.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
J. Arredondo, A. I. Chernyavsky, D. L. Jolkovsky, K. E. Pinkerton, and S. A. Grando
Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf-1/MEK1/ERK and JAK-2/STAT-3 pathways downstream of {alpha}7 nicotinic receptor in oral keratinocytes
FASEB J, October 1, 2006; 20(12): 2093 - 2101.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. J. Marks, P. Whiteaker, and A. C. Collins
Deletion of the {alpha}7, beta2, or beta4 Nicotinic Receptor Subunit Genes Identifies Highly Expressed Subtypes with Relatively Low Affinity for [3H]Epibatidine
Mol. Pharmacol., September 1, 2006; 70(3): 947 - 959.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. M. McCann, J. Bracamontes, J. H. Steinbach, and J. R. Sanes
The cholinergic antagonist {alpha}-bungarotoxin also binds and blocks a subset of GABA receptors
PNAS, March 28, 2006; 103(13): 5149 - 5154.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Y.-H. Jo, D. Wiedl, and L. W. Role
Cholinergic Modulation of Appetite-Related Synapses in Mouse Lateral Hypothalamic Slice
J. Neurosci., November 30, 2005; 25(48): 11133 - 11144.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
P. van Nierop, A. Keramidas, S. Bertrand, J. van Minnen, Y. Gouwenberg, D. Bertrand, and A. B. Smit
Identification of Molluscan Nicotinic Acetylcholine Receptor (nAChR) Subunits Involved in Formation of Cation- and Anion-Selective nAChRs
J. Neurosci., November 16, 2005; 25(46): 10617 - 10626.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. Bray, J.-H. Son, P. Kumar, and S. Meizel
Mice Deficient in CHRNA7, a Subunit of the Nicotinic Acetylcholine Receptor, Produce Sperm with Impaired Motility
Biol Reprod, October 1, 2005; 73(4): 807 - 814.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
J. Deck, S. Bibevski, T. Gnecchi-Ruscone, V. Bellina, N. Montano, and M. E. Dunlap
{alpha}7-Nicotinic acetylcholine receptor subunit is not required for parasympathetic control of the heart in the mouse
Physiol Genomics, June 16, 2005; 22(1): 86 - 92.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
A. Cormier, Y. Paas, R. Zini, J.-P. Tillement, G. Lagrue, J.-P. Changeux, and R. Grailhe
Long-Term Exposure to Nicotine Modulates the Level and Activity of Acetylcholine Receptors in White Blood Cells of Smokers and Model Mice
Mol. Pharmacol., December 1, 2004; 66(6): 1712 - 1718.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. I. Chernyavsky, J. Arredondo, L. M. Marubio, and S. A. Grando
Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes
J. Cell Sci., November 1, 2004; 117(23): 5665 - 5679.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
D. L. Herber, E. G. Severance, J. Cuevas, D. Morgan, and M. N. Gordon
Biochemical and Histochemical Evidence of Nonspecific Binding of {alpha}7nAChR Antibodies to Mouse Brain Tissue
J. Histochem. Cytochem., October 1, 2004; 52(10): 1367 - 1376.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J. Wu, A. A. George, K. M. Schroeder, L. Xu, S. Marxer-Miller, L. Lucero, and R. J. Lukas
Electrophysiological, Pharmacological, and Molecular Evidence for {alpha}7-Nicotinic Acetylcholine Receptors in Rat Midbrain Dopamine Neurons
J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 80 - 91.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. C. Hernandez, S. Vicini, Y. Xiao, M. I. Davila-Garcia, R. P. Yasuda, B. B. Wolfe, and K. J. Kellar
The Nicotinic Receptor in the Rat Pineal Gland Is an {alpha}3{beta}4 Subtype
Mol. Pharmacol., October 1, 2004; 66(4): 978 - 987.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
E. G. Severance, H. Zhang, Y. Cruz, S. Pakhlevaniants, S. H. Hadley, J. Amin, L. Wecker, C. Reed, and J. Cuevas
The {alpha}7 Nicotinic Acetylcholine Receptor Subunit Exists in Two Isoforms that Contribute to Functional Ligand-Gated Ion Channels
Mol. Pharmacol., September 1, 2004; 66(3): 420 - 429.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
O. Salminen, K. L. Murphy, J. M. McIntosh, J. Drago, M. J. Marks, A. C. Collins, and S. R. Grady
Subunit Composition and Pharmacology of Two Classes of Striatal Presynaptic Nicotinic Acetylcholine Receptors Mediating Dopamine Release in Mice
Mol. Pharmacol., June 1, 2004; 65(6): 1526 - 1535.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. Kedmi, A. L. Beaudet, and A. Orr-Urtreger
Mice lacking neuronal nicotinic acetylcholine receptor {beta}4-subunit and mice lacking both {alpha}5- and {beta}4-subunits are highly resistant to nicotine-induced seizures
Physiol Genomics, April 13, 2004; 17(2): 221 - 229.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Cui, T. K. Booker, R. S. Allen, S. R. Grady, P. Whiteaker, M. J. Marks, O. Salminen, T. Tritto, C. M. Butt, W. R. Allen, et al.
The {beta}3 Nicotinic Receptor Subunit: A Component of {alpha}-Conotoxin MII-Binding Nicotinic Acetylcholine Receptors that Modulate Dopamine Release and Related Behaviors
J. Neurosci., December 3, 2003; 23(35): 11045 - 11053.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Salas, F. Pieri, B. Fung, J. A. Dani, and M. De Biasi
Altered Anxiety-Related Responses in Mutant Mice Lacking the {beta}4 Subunit of the Nicotinic Receptor
J. Neurosci., July 16, 2003; 23(15): 6255 - 6263.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
V. Batra, A. A. Patkar, W. H. Berrettini, S. P. Weinstein, and F. T. Leone
The Genetic Determinants of Smoking
Chest, May 1, 2003; 123(5): 1730 - 1739.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
R. Salas, A. Orr-Urtreger, R. S. Broide, A. Beaudet, R. Paylor, and M. De Biasi
The Nicotinic Acetylcholine Receptor Subunit alpha 5 Mediates Short-Term Effects of Nicotine in Vivo
Mol. Pharmacol., May 1, 2003; 63(5): 1059 - 1066.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
J. R. A. Wooltorton, V. I. Pidoplichko, R. S. Broide, and J. A. Dani
Differential Desensitization and Distribution of Nicotinic Acetylcholine Receptor Subtypes in Midbrain Dopamine Areas
J. Neurosci., April 15, 2003; 23(8): 3176 - 3185.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
H. Tsuneki, R. Salas, and J. A Dani
Mouse muscle denervation increases expression of an {alpha}7 nicotinic receptor with unusual pharmacology
J. Physiol., February 15, 2003; 547(1): 169 - 179.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
L. H. Wilkins Jr., V. P. Grinevich, J. T. Ayers, P. A. Crooks, and L. P. Dwoskin
N-n-Alkylnicotinium Analogs, a Novel Class of Nicotinic Receptor Antagonists: Interaction with alpha 4beta 2* and alpha 7* Neuronal Nicotinic Receptors
J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 400 - 410.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
A Bradaia and J Trouslard
Fast synaptic transmission mediated by {alpha}-bungarotoxin-sensitive nicotinic acetylcholine receptors in lamina X neurones of neonatal rat spinal cord
J. Physiol., November 1, 2002; 544(3): 727 - 739.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
J. Arredondo, V. T. Nguyen, A. I. Chernyavsky, D. Bercovich, A. Orr-Urtreger, W. Kummer, K. Lips, D. E. Vetter, and S. A. Grando
Central role of {alpha}7 nicotinic receptor in differentiation of the stratified squamous epithelium
J. Cell Biol., October 28, 2002; 159(2): 325 - 336.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. Bray, J.-H. Son, and S. Meizel
A Nicotinic Acetylcholine Receptor Is Involved in the Acrosome Reaction of Human Sperm Initiated by Recombinant Human ZP3
Biol Reprod, September 1, 2002; 67(3): 782 - 788.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. B. Levy and C. Aoki
alpha 7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex
J. Neurosci., June 15, 2002; 22(12): 5001 - 5015.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
R. S. Broide, R. Salas, D. Ji, R. Paylor, J. W. Patrick, J. A. Dani, and M. De Biasi
Increased Sensitivity to Nicotine-Induced Seizures in Mice Expressing the L250T alpha 7 Nicotinic Acetylcholine Receptor Mutation
Mol. Pharmacol., March 1, 2002; 61(3): 695 - 705.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
R. A. Pierce and N. M. Nguyen
Prenatal Nicotine Exposure and Abnormal Lung Function
Am. J. Respir. Cell Mol. Biol., January 1, 2002; 26(1): 10 - 13.
[Full Text] [PDF]

Home page
 JGPHome page
M. E. Nelson, F. Wang, A. Kuryatov, C. H. Choi, V. Gerzanich, and J. Lindstrom
Functional Properties of Human Nicotinic Achrs Expressed by Imr-32 Neuroblastoma Cells Resemble Those of {alpha}3{beta}4 Achrs Expressed in Permanently Transfected Hek Cells
J. Gen. Physiol., November 1, 2001; 118(5): 563 - 582.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Fabian-Fine, P. Skehel, M. L. Errington, H. A. Davies, E. Sher, M. G. Stewart, and A. Fine
Ultrastructural Distribution of the {alpha}7 Nicotinic Acetylcholine Receptor Subunit in Rat Hippocampus
J. Neurosci., October 15, 2001; 21(20): 7993 - 8003.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
L. P. Dwoskin and P. A. Crooks
Competitive Neuronal Nicotinic Receptor Antagonists: A New Direction for Drug Discovery
J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 395 - 402.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. Klink, A. d. K. d'Exaerde, M. Zoli, and J.-P. Changeux
Molecular and Physiological Diversity of Nicotinic Acetylcholine Receptors in the Midbrain Dopaminergic Nuclei
J. Neurosci., March 1, 2001; 21(5): 1452 - 1463.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Cordero-Erausquin and J.-P. Changeux
Tonic nicotinic modulation of serotoninergic transmission in the spinal cord
PNAS, February 15, 2001; (2001) 41600698.
[Abstract] [Full Text]

Home page
 J. Pharmacol. Exp. Ther.Home page
A. J. Grottick, G. Trube, W. A. Corrigall, J. Huwyler, P. Malherbe, R. Wyler, and G. A. Higgins
Evidence That Nicotinic alpha 7 Receptors Are Not Involved in the Hyperlocomotor and Rewarding Effects of Nicotine
J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 1112 - 1119.
[Abstract] [Full Text]

Home page
 J. Neurosci.Home page
S. A. Ross, J. Y. F. Wong, J. J. Clifford, A. Kinsella, J. S. Massalas, M. K. Horne, I. E. Scheffer, I. Kola, J. L. Waddington, S. F. Berkovic, et al.
Phenotypic Characterization of an alpha 4 Neuronal Nicotinic Acetylcholine Receptor Subunit Knock-Out Mouse
J. Neurosci., September 1, 2000; 20(17): 6431 - 6441.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
R. W. Oppenheim, D. Prevette, A. D'Costa, S. Wang, L. J. Houenou, and J. M. McIntosh
Reduction of Neuromuscular Activity Is Required for the Rescue of Motoneurons from Naturally Occurring Cell Death by Nicotinic-Blocking Agents
J. Neurosci., August 15, 2000; 20(16): 6117 - 6124.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
D. Ji and J. A. Dani
Inhibition and Disinhibition of Pyramidal Neurons by Activation of Nicotinic Receptors on Hippocampal Interneurons
J Neurophysiol, May 1, 2000; 83(5): 2682 - 2690.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. T. Dineley and J. W. Patrick
Amino Acid Determinants of alpha 7 Nicotinic Acetylcholine Receptor Surface Expression
J. Biol. Chem., April 28, 2000; 275(18): 13974 - 13985.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Lena, A. de Kerchove d'Exaerde, M. Cordero-Erausquin, N. Le Novere, M. del Mar Arroyo-Jimenez, and J.-P. Changeux
Diversity and distribution of nicotinic acetylcholine receptors in the locus ceruleus neurons
PNAS, October 12, 1999; 96(21): 12126 - 12131.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
M. d. M. Arroyo-Jimenez, J.-P. Bourgeois, L. M. Marubio, A.-M. Le Sourd, O. P. Ottersen, E. Rinvik, A. Fairen, and J.-P. Changeux
Ultrastructural Localization of the alpha 4-Subunit of the Neuronal Acetylcholine Nicotinic Receptor in the Rat Substantia Nigra
J. Neurosci., August 1, 1999; 19(15): 6475 - 6487.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
R. A. Cardoso, S. J. Brozowski, L. E. Chavez-Noriega, M. Harpold, C. F. Valenzuela, and R. A. Harris
Effects of Ethanol on Recombinant Human Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes
J. Pharmacol. Exp. Ther., May 1, 1999; 289(2): 774 - 780.
[Abstract] [Full Text]

Home page
 NeuroscientistHome page
M. R. Picciotto
Nicotine Addiction: From Molecules to Behavior
Neuroscientist, November 1, 1998; 4(6): 391 - 394.
[Abstract] [PDF]

Home page
 J. Neurosci.Home page
J. Kehoe and J. M. McIntosh
Two Distinct Nicotinic Receptors, One Pharmacologically Similar to the Vertebrate alpha 7-Containing Receptor, Mediate Cl Currents in Aplysia Neurons
J. Neurosci., October 15, 1998; 18(20): 8198 - 8213.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. J. Frazier, A. V. Buhler, J. L. Weiner, and T. V. Dunwiddie
Synaptic Potentials Mediated via alpha -Bungarotoxin-Sensitive Nicotinic Acetylcholine Receptors in Rat Hippocampal Interneurons
J. Neurosci., October 15, 1998; 18(20): 8228 - 8235.
[Abstract] [Full Text] [PDF]

Home page
 Learn. Mem.Home page
R. Paylor, M. Nguyen, J. N. Crawley, J. Patrick, A. Beaudet, and A. Orr-Urtreger
alpha 7 Nicotinic Receptor Subunits Are Not Necessary for Hippocampal-Dependent Learning or Sensorimotor Gating: A Behavioral Characterization of Acra7-Deficient Mice
Learn. Mem., September 1, 1998; 5(4): 302 - 316.
[Abstract] [Full Text]

Home page
 J. Neurosci.Home page
M. Zoli, C. Lena, M. R. Picciotto, and J.-P. Changeux
Identification of Four Classes of Brain Nicotinic Receptors Using beta 2 Mutant Mice
J. Neurosci., June 15, 1998; 18(12): 4461 - 4472.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Cordero-Erausquin and J.-P. Changeux
Tonic nicotinic modulation of serotoninergic transmission in the spinal cord
PNAS, February 27, 2001; 98(5): 2803 - 2807.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an eLetter
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (241)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Orr-Urtreger, A.
Right arrow Articles by Beaudet, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Orr-Urtreger, A.
Right arrow Articles by Beaudet, A. L.

-
-

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help
-
Copyright 2009 by Society for Neuroscience ONLINE ISSN: 1529-2401
-