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. 2000 Oct 10;97(21):11575-80.
doi: 10.1073/pnas.97.21.11575.

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice

S Shen  1 , C Spratt, W J Sheward, I Kallo, K West, C F Morrison, C W Coen, H M Marston, A J Harmar
Affiliations

Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice

S Shen et al. Proc Natl Acad Sci U S A. .

Abstract

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone. Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function. VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner. To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct. The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose. High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas. Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD). These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock.

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Figures

Figure 1
Figure 1
Two-step genetic manipulation of YAC DNA by homologous recombination (a) using YAC amplification vector pYAM4 (b) and YAC insertion vector pYIV3 (c). A YAC contains an insert of genomic DNA between short and long vector arms. In step 1, genomic sequences flanking the desired site of insertion in the target gene are cloned either side of the HA-IRES-lacZ-ADE2 cassette in pYIV3. Integration of the HA-IRES-lacZ-ADE2 cassette into the YAC can be verified by hybridization with an ADE2 probe. In step 2, pYAM4 is used to replace the long vector arm so that the YAC DNA can be amplified when grown in medium containing galactose.
Figure 2
Figure 2
Ethidium bromide staining of pulsed-field gels showing YAC DNA amplification and purification. Sizes in kilobases of yeast chromosomes are indicated, and bands corresponding to YAC DNA are indicated with arrowheads. (a and b) YAC clones before amplification, run on a 0.9% agarose gel in 0.5× TBE, at 6V/cm, 4°C for 24 h with 60 s switch time. (a) DNA from yeast containing a 400-kb YAC; (b) DNA from the 550-kb YAC HSC7E526. The intensity of ethidium bromide staining of the 550 kb YAC (b) is approximately half that of the band above, which contains two yeast chromosomes (555 and 610 kb) unresolved from one another. (c) DNA from pYIV3- and pYAM4-modified HSC7E526, run on a preparative scale in a 1% agarose gel in 0.25× TAE, at 6V/cm, 4°C for 32 h with 30 and 55 s switch time. Thirty-nine agarose plugs (0.5 cm × 0.15 cm × 1 cm) containing YAC DNA were loaded into a sample well 19.5 cm wide and sealed with agarose. After electrophoresis, the central 17 cm of the gel was kept for the purification of YAC DNA, and the remainder of the gel was stained with ethidium bromide and photographed. The two stained portions of the gel, each containing about 0.5 cm of the preparative lane, are shown. The intensity of staining of the YAC DNA is 1.5 to ≈2× that of the band above. (d) Gel-purified YAC DNA before microinjection into fertilized eggs.
Figure 3
Figure 3
Histochemical (A–J) and immunocytochemical (K–O) demonstration of transgene expression in brain and peripheral tissues of A108.2 transgenic mice. (A) Complete coronal slice of brain showing lacZ expression in the SCN and in the medial habenula (MHb). Cells positive for lacZ (B) in the glomerular and internal granular layers of the olfactory bulb, (C) in the lateral septum, (D) in a small number of cells in the medial preoptic area (MPO) and in the supraoptic nuclei (SON), (E) in the SCN, (F) in the ependymal cell layer of the rostral portion of the third ventricle (III) and in the walls of numerous blood vessels (arrowed), (G) in the ependymal cell layer of the fourth ventricle (IV), (H) in a slice of the brain that includes both the midbrain and the caudal portion of the forebrain, showing strong expression of lacZ in the walls of the major cerebral blood vessels, (I) in the pancreas of a transgenic (tg), but not of a wild-type (wt) mouse, and (J) in the spleen of a transgenic (tg), but not of a wild-type (wt) mouse. β-galactosidase-like immunoreactivity (K) in cells and processes in the SCN and (L) in the medial habenular nucleus. HA-like immunoreactivity (M and N) in the medial habenular nucleus; the membrane-associated immunoreactivity for this antigen is clearly seen in some cases (arrowed). Labeling was absent (O) when the antiserum had been preincubated with excess HA peptide. lv, lateral ventricle; Aq, aqueduct; LSi, intermediate portion of the lateral septal nucleus; LSv, ventral portion of the lateral septal nucleus; ox, optic chiasm.
Figure 4
Figure 4
Representative profiles of locomotor activity in VPAC2R transgenic and wild-type mice. Records are double-plotted so that 48 h are shown for each horizontal trace and consecutive days are aligned vertically and duplicated diagonally. Periods of darkness are shaded. For the first 6 days, animals were exposed to a 12:12 h light:dark (LD) cycle (dark from 19:00 to 07:00). On day 7, the LD cycle was advanced by 8 h (dark from 11:00 to 23:00) via a shortened light period of four hours. On day 17, the LD cycle was returned to the original lighting regime (dark 19:00 to 07:00) via a light period of 20 h. From day 30, animals were maintained in constant darkness.
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References

    1. Mikkelsen J D, Fahrenkrug J. Brain Res. 1994;656:95–107. - PubMed
    1. Loren I, Emson P C, Fahrenkrug J, Bjorklund A, Alumets J, Hakanson R, Sundler F. Neuroscience. 1979;4:1953–1976. - PubMed
    1. Tanaka M, Ichitani Y, Okamura H, Tanaka Y, Ibata Y. Brain Res Bull. 1993;31:637–640. - PubMed
    1. Ibata Y, Tanaka M, Ichitani Y, Takahashi Y, Okamura H. Neuroreport. 1993;4:128–130. - PubMed
    1. Shinohara K, Tominaga K, Isobe Y, Inouye S T. J Neurosci. 1993;13:793–800. - PMC - PubMed

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