Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat.

Abstract

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wildtype vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.Since its discovery in 1961 the diabetes insipidus Brattleboro rat (phenotype, di/di) with impaired vasopressin (VP) synthesis has become an extremely useful model for many disciplines to study hypothalamic diabetes insipidus and to clarify problems related to the physiology ofVP (1). The di/di rat exhibits a recessively inherited hypothalamic diabetes insipidus due to a single-base deletion in the unique VP gene (2). This point mutation results in the synthesis of an altered VP precursor whose C-terminal glycopeptide (GP) moiety, because of a frame-shift, is replaced by a nonglycosylated amino acid sequence (2). As a consequence intracellular processing and axonal transport toward the neural lobe ofthe mutant precursor are blocked. Paradoxically, a small number of solitary hypothalamic neurons seems to express wild-type VP gene products that are transported toward the neural lobe (3-5). To explain these results, an intrachromosomal gene conversion between the homologous exons of the VP and oxytocin (OT) genes has been proposed (6). To (termed, di/+) and that the number of these neurons increases linearly with age. The findings imply that genomic alterations occur after mitotic division has ceased and are compatible with the intrachromosomal gene conversion within the VP/OT gene locus. MATERIALS AND METHODSAnimals and Fixation. Male and female di/di Brattleboro rats of 18 days to 79 weeks of age were obtained from Harlan (Zeist, The Netherlands) or homebred. They were individually checked for water consumption and diuresis. The animals were anaesthetized with Nembutal (60 mg/kg, i.p.) and perfused intracardially with 0.9% NaCl (shortly) and then with 0.1 M sodium phosphate-buffered 4% (wt/vol) paraformaldehyde (pH 7.4) and pieces of tissue then wer...

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