Ethanol hypersensitivity and olfactory discrimination defect in mice lacking a homolog of Drosophila neuralized

Abstract

Neurogenic genes in the Notch receptor-mediated signaling pathway play important roles in neuronal cell fate specification as well as neuronal differentiation. The Drosophila neuralized gene is one of the neurogenic genes. We have cloned a mouse homolog of Drosophila neuralized, m-neu1, and found that the m-neu1 transcript is expressed in differentiated neurons. Mice deficient for m-neu1 are viable and morphologically normal, but exhibit specific defects in olfactory discrimination and hypersensitivity to ethanol. These findings reveal an essential role of m-neu1 in ensuring proper processing of certain information in the adult brain.

Figure 1

The predicted amino acid sequence of M-Neu1 and sequence alignment of M-Neu1 and D-Neu. The first methionine of M-Neu1 was selected based on Kozak's rule and identification of an in-frame upstream termination codon. The amino acid sequences of M-Neu1 and D-Neu were aligned by using the CLUSTAL W sequence alignment program. Identical residues are outlined and shaded, and similar residues are outlined. − represents gap in the sequence. The sequences of the C3HC4 RING zinc finger domain are underlined.

Figure 2

Tissue distribution and expression profile of m-neu1 mRNA. (a) RT-PCR analysis of m-neu1 expression during mouse embryogenesis. Whole embryos were isolated from embryonic day 7.5 (E7.5) to embryonic day 17.5 (E17.5) and used to prepare total RNA. An equivalent amount of total RNA was transcribed and subjected to PCR. RNAs treated without reverse transcriptase (RT−) were used as negative controls. (b) Northern blot analysis of m-neu1 expression in adult mouse organs. Two micrograms of poly(A)⁺ RNA was used in each lane. A probe against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.

Figure 3

m-neu1 expression in embryonic, postnatal, and adult mice. Whole embryos or sections were hybridized with an antisense RNA probe for m-neu1. (a) m-neu1 expression in an E8.5 embryo. High levels of m-neu1 expression in E8.5 embryo are observed in the forebrain neural fold (f), the hindbrain neural fold (h), the first branchial arch (b), the neural tube (n), and the somites (s). (b) m-neu1 expression in E13.5 olfactory epithelium (oe) and vomeronasal organ (vno). (c) m-neu1 expression in P0 cerebellum. (d) m-neu1 expression in adult brain. High levels of m-neu1 expression in adult brain are observed in the cerebral cortex (cc), cerebellum (cb), striatum (s), hippocampus (h), and dentate gyrus (d). (e) Cytoplasmic localization of M-Neu1/GFP fusion protein. A construct of the m-neu1 gene fused to the gene encoded the green fluorescence protein was transfected into cultured mouse neuroblastoma Neuro2a cells (America Type Culture Collection). The expressed fusion protein is primarily in the cytoplasm.

Figure 4

Targeted disruption of the m-neu1 gene. (a) Schematic diagram of the targeting strategy, showing the organization of the wild-type m-neu1 genomic region, the targeting construct, and the mutant allele that results from homologous recombination of the targeting vector at the m-neu1 locus. Exons are shown as closed boxes; neo, neomycin-resistant gene cassette; tk, thymidine kinase gene cassette. The transcription orientation of neo cassette is shown by the arrow. The 5′ and 3′ probes that are external to the vector region are indicated at the bottom. (b) Southern blot analysis of BamHI-digested DNAs from six littermates after hybridization with a 5′ probe. The wild-type allele yields a 16-kb band, and the mutant allele yields a 10-kb band. Genotypes are indicated above each lane: wild-type (+/+), heterozygous (+/−), and homozygous (−/−). (c) Genotype analysis by PCR. Three primers were used (see Methods). Primer 1 and primer 2 generate a 190-bp fragment that represents wild-type allele, whereas primer 2 and primer 3 produce a 270-bp fragment indicative of the knockout allele. (d) Detection of m-neu1 transcript by Northern blot analysis. Brain poly(A)⁺ RNAs (2 μg) from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) were blot-hybridized with a probe specific for m-neu1. A β-actin probe was used as a control.

Figure 5

Adult neurogenesis in the dentate gyrus and chain migration of neuronal precursors in the rostral migratory stream (RMS) are indistinguishable in wild-type mice and m-neu1^−/− mice. BrdUrd-labeled neurons (arrow) in the dentate gyrus of wild-type mice (a) and m-neu1^−/− mice (b). BrdUrd-labeled neuronal precursors (arrowhead) in the rostral migratory stream of wild-type (c) and m-neu1^−/− mice (d).

Figure 6

m-neu1^−/− mice are defective in olfactory discrimination. The preference (%) shown on the y axis is the percentage of isovaleric acid solution consumed of the total solution consumed. Values represent group means ± SD. Data analysis (one-way ANOVA) revealed the significant difference (P < 0.05) between wild-type (n = 13) and m-neu1^−/− mice (n = 16) at the 10⁻³ M, 10⁻⁴ M, and 10⁻⁵ M isovaleric acid concentrations.

Figure 7

m-neu1^−/− mice exhibit normal learning and memory in the Morris water maze. (a) Escape latencies are comparable for the m-neu1^−/− mice and wild-type mice in the acquisition phase. The time it took for the mouse to reach the platform was plotted for the visible-platform task in the first 2 days (blocks 1–4) and the hidden-platform task in the following 5 days (blocks 5–14). (b) Quadrant preferences of m-neu1^−/− mice and wild-type mice in the probe test, which is just after the 7-day training and is in the absence of platform. The time spent in each quadrant during a 60-s probe test is plotted. There were no significant differences between m-neu1^−/− mice and wild-type mice, indicating that m-neu1^−/− mice have normal memory. All values are mean ± SEM.

Figure 8

m-neu1^−/− mice are hypersensitive to the effect of ethanol on motor coordination. Wild-type and m-neu1^−/− mice were injected with ethanol (2.0 g/kg, i.p.), and their performance on the rotarod was examined 30, 40, and 50 min after ethanol injection. By 50 min, all of the wild-type mice stayed on the rotarod longer than 150 s, and their latency was recorded as 150 s. Values represent group mean ± SD. Data analysis (one-way ANOVA) revealed the significant difference (P < 0.05) between wild-type (n = 15) and m-neu1^−/− (n = 16) mice at each time point tested.