Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents

Abstract

The alpha7 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 alpha7 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 alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 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.

Fig. 1.

Gene targeting of the neuronal nAChR α7 subunit. a, Partial genomic structure of the murine α7 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 andInt-2) to confirm the deletion. The targeting vector was used to obtain a replacement mutation and contains a neomycin resistance gene (neo^r) 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 shownbelow the targeting vector. b, Southern blot analysis identifies the α7 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 α7 gene expression in brains of (+/+), (+/−), and (−/−) animals, using the Acrα7 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 α7 protein (Chen and Patrick, 1997) were used.

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.

Fig. 3.

Nicotine and α-BGT binding in mutant mice. Autoradiography was performed with [³H]nicotine and [¹²⁵I]α-BGT as described in Materials and Methods with +/+ mice shown on the left and −/− shown on the right. The panels marked coldα-BGT included excess nonradioactive α-BGT as competitor.

Fig. 4.

Nicotine induces currents in control but not in α7 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). Thesolid 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 α7 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 α7 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 α7 null (−/−) mice (4 animals, 35 cells). n, Number of cells recorded; I, current in pA.