Human Molecular Genetics, 2000, Vol. 9, No. 20 3101-3110
© 2000 Oxford University Press
Disruption of the mouse Necdin gene results in hypothalamic and behavioral alterations reminiscent of the human Prader–Willi syndrome
INSERM U491/IBDM, Faculté de Médecine, 27 Boulevard Jean Moulin, F-13385 Marseille Cedex 5, France, 1INSERM U259, Laboratoire des Comportements Adaptatifs, Institut François Magendie, Rue Camille Saint-Saëns, F-33077 Bordeaux, France and 2LGPD/IBDM/Université de Méditerranée, Campus de Luminy, Marseille Cedex 8, France
Received 2 October 2000; Revised and Accepted 9 October 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Prader–Willi syndrome (PWS) is a complex neurogenetic disorder with considerable clinical variability that is thought in large part to be the result of a hypothalamic defect. PWS results from the absence of paternal expression of imprinted genes localized in the 15q11–q13 region; however, none of the characterized genes has so far been shown to be involved in the etiology of PWS. Here, we provide a detailed investigation of a mouse model deficient for Necdin. Linked to the mutation, a neonatal lethality of variable penetrance is observed. Viable Necdin mutants show a reduction in both oxytocin-producing and luteinizing hormone-releasing hormone (LHRH)-producing neurons in hypothalamus. This represents the first evidence of a hypothalamic deficiency in a mouse model of PWS. Necdin-deficient mice also display increased skin scraping activity in the open field test and improved spatial learning and memory in the Morris water maze. The latter features are reminiscent of the skin picking and improved spatial memory that are characteristics of the PWS phenotype. These striking parallels in hypothalamic structure, emotional and cognitive-related behaviors strongly suggest that NECDIN is responsible for at least a subset of the multiple clinical manifestations of PWS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Prader–Willi syndrome (PWS) is a neurogenetic disorder which occurs in 1 in 10 000–15 000 births (1). Diagnosis for this disease is based on a scoring system established from a list of
20 major and minor criteria, differing in infancy from those in child- and adulthood (2) (OMIM 176270). The major findings include a transient infantile hypotonia and a failure to thrive during the newborn period. From 2 years of age, PWS individuals display hyperphagia leading to severe obesity. They also present a global developmental delay, typical facial features, hypogonadotropic hypogonadism, mild to moderate mental retardation, learning profiles with particular strengths and weaknesses and characteristic behavioral problems. Furthermore, a variety of less frequent findings have been described, for example respiratory distress at birth (3). Thus, PWS is a complex disorder with considerable clinical variability (1). Many of the manifestations are thought to be due to hypothalamic deficiency (1,4). PWS is a classical example of a human disease that reveals the regulation of the 15q11–q13 chromosomal region by genomic imprinting, a process by which a subset of autosomal genes is differentially expressed depending on parent of origin. All of the PWS patients examined have genetic abnormalities (large paternal deletion, maternal disomy or imprinting mutation) resulting in the inactivation of paternally expressed genes. Furthermore, there is no reported case of a normal paternal copy of 15q11–q13 with an inheritance consistent with a single mutated gene. These data imply that PWS is a multigenic syndrome (5–8).
The identification of genes involved in PWS is difficult, requiring the isolation of candidate genes among all the paternally expressed sequences in a region spanning >1.5 Mb. One approach is to characterize all the human candidates in the region and to clone their mouse orthologs. This allows the creation of animal models in order to determine the role of each gene in the etiology of PWS (8).
Is the mouse an appropriate model for PWS? The mouse 7C chromosomal region is a region of conserved synteny to the human chromosome 15q11–q13 region (9). Four mouse models have so far been reported as Prader–Willi models, resulting in the deficiency of paternal gene expression in the 7C chromosomal (10–13). The phenotype observed is lethality during the first post-natal week, associated with poor feeding (10,11) or postnatal lethality before weaning associated with respiratory distress (12) or hypotonia, growth retardation and lethality in 80% of newborn mice (13). Based only on these data, these mouse phenotypes were correlated with the feeding difficulties and failure to thrive that characterize PWS infants. Furthermore, such models do not allow the discrimination of the role of each single gene in the PWS phenotype.
Six imprinted genes have so far been described in humans (Fig. 1a): SNURF-SNRPN (14), ZNF127 (15), IPW (16), MAGEL2 (17) and NDN (18,19) (Fig. 1a). All of these genes have orthologs in the mouse 7C chromosomal region, with the same imprinted status. Mice deficient for Snrpn (11), Snurf (13), Zfp127 (20) and Ipw (20) have been independently created but no role in the etiology of PWS could be ascribed to any of these genes. Two lines of Ndn-deficient mice have been described. In one model, an early postnatal lethality probably due to respiratory distress was observed, although the penetrance of this mutation was highly variable depending on the genetic background (21). The second model was presented only as viable, fertile without any sign of obesity (22).
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As described above, the features of PWS are complex. However, none of the mouse models has been studied for subtle morphological modifications or changes in behavior.
The human NDN gene is a strong candidate to be involved in the etiology of PWS, due to its expression in the nervous system and the observation that it is inactivated in PWS patients (19). To investigate the biological function of Necdin and its role in the etiology of PWS, we generated Ndn-deficient mice and performed a detailed histological and behavioral analysis. Our study revealed strong parallels in terms of morphological, behavioral and cognitive modifications, between the effects of Ndn mutation in mice and key features of PWS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Generation of Ndn-deficient mice
To investigate the in vivo function of Necdin, we inactivated the Ndn gene in the mouse germ line by conventional gene targeting (Fig. 1b). The promoter and the first two-thirds of the Ndn coding sequence were replaced with the HSV-TK-Neo cassette flanked by loxP sites, which allows subsequent CRE-dependent deletion of the marker gene. Chimeric males, derived from three independent recombinant embryonic stem (ES) cell clones, were mated with either wild-type C57Bl6/J females or C57Bl6/J females expressing CRE recombinase (23). Two lines of mutant N1 progeny were thereby produced. In the first one, the Ndn paternal allele was replaced with Neo (+Neo), which enabled us to study the imprinting state of Neo in this genetic context. In the second line, the Neo marker was deleted, in order to investigate the consequences of Ndn deficiency in the absence of putative interference between the Neo cassette and the normal transcription of neighboring genes (24).
In both lines, matings between Ndn heterozygotes produced N2 progenies of wild-type, heterozygous (+m/–p or –m/+p) and homozygous mutant (–/–) genotypes as determined by genomic Southern blot analysis (Fig. 2a). Northern blot analysis (Fig. 2b and c) or RT–PCR analysis (Fig. 3a) was performed to verify the complete absence of Ndn transcripts in homozygous mutants as well as heterozygous animals in which the paternal allele was deleted (–m/–p and +m/–p). In addition, these analyses demonstrated the complete absence of Ndn expression from the maternal allele in the neonatal or adult brain as well as in other tissues (data not shown). The absence of Ndn expression was also verified at the protein level by western blot analysis on whole brain homogenates (Fig. 2d), using a peptide-specific antibody against the C-terminal region of Necdin.
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The imprinting state of Neo was studied using RT–PCR on RNA extracted from mouse brains with a maternally or paternally derived Neo allele (Fig. 3a). In no case were Neo transcripts detected. Such a transcriptional inhibition of Neo could result from the presence of the viral HSV-TK promoter, which might be inactive in this context. Alternatively, regulatory elements of the Ndn promoter could be necessary to allow an active brain transcription (25) or an imprinting control of this locus. Both hypotheses are supported by the results obtained by Gérard et al. (21). These authors disrupted the Ndn gene by replacing exclusively the coding part with the PGK-Neo cassette and found that PGK-Neo is imprinted, being expressed exclusively from the paternal allele.
To generate Necdin mutant mice, mice in which the Neo marker was entirely deleted, chimeric males were crossed with C57Bl6/J females expressing CRE in the oocyte to produce the N1 generation. In this generation 34.5% instead of the expected Ndn 50% mutants were identified. A second backcross between N1 +m/–p males and C57Bl6 wild-type females produced N2 offspring. Genotyping revealed 18% mutants in this generation (expected 50%; Table 1). In both generations, lack of mutants was observed in males and females in equal proportions. The observed ratio was estimated after genotyping at weaning. To investigate whether the lethality due to Ndn deficiency is a pre- or postnatal event, we genotyped 40 mice 2 h after birth. At this time point we observed a 50% ratio between mutant and wild-type mice, suggesting that lethality occurred postnatally. In addition, we followed a cohort of 24 newborn mice over a 48 h interval and found that five animals died during this period; they appeared cyanotic and showed signs of respiratory distress. This phenotype appears similar to the phenotype described by Gérard et al. (21). In conclusion, we observed a partial early postnatal lethality due to the Ndn mutation. Furthermore, the penetrance of this phenotype is apparently increased in the C57Bl6/J background. Surviving mice lacking Necdin expression were viable, fertile and show no sign of obesity until 18 months of age, the latest time point observed. Dissection of paternal-deficient and wild-type mice revealed no gross morphological abnormalities.
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To check that the introduced mutation has no influence on the genomic environment, we analyzed the integrity of transcription of neighboring genes by RT–PCR. This analysis revealed that Snrpn, Zfp127 and Magel2 (a member of the Mage/Ndn gene family) (Figs 1a and 3b) show no differences in their transcription patterns, as expected.
In the following studies, the analyses of Ndn mutants were performed on an N2 population of adult male mice issued from the following backcross: [+m/–p male N1 (chimeric 129SV–C57Bl6/J-CRE) x wild-type female C57Bl6/J] and compared paternally deficient mice for Ndn (+m/–p) with wild-type littermates (+m/+p). We chose this population in order to avoid interference with the female reproductive cycle and to get all the results on the same genetic background as used for the behavioral studies. Due to the strong influence of the genetic strain background on mouse behaviors such as learning, aggression and anxiety, we followed the recommendations concerning the genetic background on behavioral analysis (26).
Ndn is expressed in a subpopulation of postmitotic neurons in newborn and adult brain
PWS patients show no obvious abnormalities in brain morphology (27). In accord with this finding, general histological markers, like Nissl blue staining and MAP2 immunolabeling on brain sections, revealed no obvious differences in brain structure in Ndn mutant mice (data not shown).
In order to focus the analysis of Ndn-deficient mice on specific brain structures, we first performed in situ hybridization histochemistry (ISHH) using a specific probe to the 3' untranslated region (3'-UTR) of Ndn on wild-type mouse brain. We found that Ndn expression was restricted to postmitotic neurons in specific regions of the adult brain. For example, a strong expression is detected in the locus coeruleus, whereas no neurons were labeled in the cerebellar cortex (Fig. 4a). Importantly, even in the hypothalamus, where general expression is the highest, Ndn is not expressed in all postmitotic neurons (Fig. 4b and c).
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In conclusion, we find that the expression pattern of Ndn defines regionally distinct subpopulations of neurons in adult brain; therefore, Ndn is not a pan-neuronal marker of postmitotic neurons, as previously reported (28).
The number of hypothalamic oxytocin- and luteinizing hormone-releasing hormone (LHRH)-producing neurons is decreased in Ndn mutant mice
Hypothalamic deficiencies have been suggested to underlie a number of symptoms in PWS (4). In this regard, we found that Ndn is highly expressed in this area during embryogenesis and in adulthood (A. Massacrier et al., in preparation). Histological examination of the hypothalamic structure in adult Ndn (+m/–p) mutant mice revealed no obvious abnormalities (data not shown). However, since Ndn is expressed only in a subpopulation of postmitotic neurons, subtle changes could be expected.
In PWS patients, a significantly reduced number of oxytocin-expressing neurons was described in the paraventricular nucleus (PVN) (4). To investigate this cell population in Ndn mutant mice we counted oxytocin-producing neurons in consecutive sections of the hypothalamus (Fig. 5a). In lateral parts of the PVN (Fig. 5b and c) we observed a significant decrease of 29% in the number of oxytocin-expressing neurons (Ndn +m/–p: 224.75 ± 16, mean ± SD, n = 4; wild-type: 313 ± 45.4, n = 4; P = 0.009).
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In PWS patients, alterations in LHRH neurons are thought to be responsible for the decreased levels of sex hormones, resulting in non-descended testes and undersized sex organs (4). LHRH neurons are consistently found within the preoptic area (POA) where Ndn is highly expressed (Figs 4c–f and 5d). Using double labeling (ISHH for Ndn and immunocytochemistry for LHRH), we demonstrated cellular colocalization of Ndn mRNA and LHRH protein expression (Fig. 5e–g). Based on the overall distribution of LHRH cells in the forebrain of the adult rat (29), we counted all the LHRH neurons located within a 1.5 mm span covering the medial POA (MPOA), the region containing the majority of these cells (Fig. 5a and d). LHRH neurons were identified using an anti-LHRH (LR1) antibody (Fig. 5d and e), allowing the detection of both types of LHRH cell. Counting of all labeled neurons on consecutive sections revealed a statistically significant loss of
25% of LHRH neurons in the Ndn mutants compared with the wild-type (knockout +m/–p: 205.8 ± 32, n = 7; wild-type: 276.7 ± 49, n = 7; P = 0.0163).
Behavioral studies
Besides a motor delay, PWS patients present behavioral disorders often related to emotional problems like temper tantrums, stubbornness, obsessive–compulsive characteristics and difficulty in adapting to a change in routine (30,31). Furthermore, up to 95% of the patients manifest a skin picking behavior resulting from incessant scraping (30,32). PWS patients exhibit cognitive impairments (most patients are mildly retarded) with, however, some strengths in specific cognitive patterns. Typically, a relative strength in reading, particular visual–spatial skills, excellent long-term memory as well as a common unusual skill to perform jigsaw puzzles have been described. Weaknesses have been observed in arithmetic, sequential processing and short-term memory (1,31,33,34).
To investigate whether Ndn-deficiency is associated with behavioral changes resembling the alterations found in PWS patients, we studied motor capacities, emotional behavior and cognitive functions in paternal deficiency (N2 males +m/–p, n = 14) and wild-type littermates (+m/+p, n = 18).
Spontaneous behavior: skin scraping is increased in Ndn mutant mice.
The Ndn +m/–p mutant mice show normal motor-related behavior as measured in the inclined plane, beam balance test and the extension reflex task (data not shown). Furthermore, the Ndn +m/–p mice were not different from wild-type mice in their daytime activity measured in circular alleys (Fig. 6a) [F(1,30) = 2.80, P > 0.05], in the elevated plus maze test [analysis performed for the time spent in the open and closed arms: F(1,29) = 1.65, P > 0.05] and in the open field (Fig. 6b) (P > 0.05). These data also indicated that locomotion–exploratory activity was not modified.
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In the elevated plus maze test (Fig. 6c), wild-type and mutant mice spent the same amount of time in the closed and open arms [interaction arm x group effect: F(1,29) = 0.339, P < 0.05], indicating that the anxiety level was not modified; the index of anxiety levels was not different among both groups (P > 0.05). Initial latency was also not different among groups (P < 0.05).
In the open field test, fear-associated parameters (faeces, urination and toilets) (Fig. 6d) were not altered by the mutation (P > 0.05) with the exception, however, of the latency to quit the first central square which was higher in mutant mice compared with the wild-type (P < 0.05). Interestingly, skin scraping was significantly increased in paternal-deficient mice (P < 0.01) (Fig. 6d). This characteristic observed in Ndn-deficient mice could be seen as analogous to the skin-picking behavior described in PWS patients.
Cognitive function: spatial learning is improved in Ndn mutant mice.
Spatial learning was assessed in the Morris water maze (MWM) in which animals have to locate a hidden platform using spatial cues. This paradigm was used since visual processing tasks and long-term memory, two parameters that are tested in the water maze, are well described aspects of the cognitive profile of PWS patients (2,31). After 9 days of training, both groups of mice improved their performance as indicated by the decreasing escape latencies [days effect: F(8,240) = 11.36, P < 0.001] (Fig. 7a) and the decreasing path length (data not shown) [days effect: F(8,240) = 12.57, P < 0.001] over the course of training. However, there were significant differences in the rate of acquisition between groups. The latency to find the hidden platform [F(1,30) = 4.38, P < 0.05] and the distance covered during the search was reduced (data not shown) [F(1,30) = 4.75, P < 0.05] were significantly decreased in the Ndn mutants mice compared with the wild-type. The observation that both groups of mice showed comparable behavioral performances during the first days of testing indicates that differences in learning were not related to differences in emotional status. In the probe trial, given on the 7th day of testing (before training), the Ndn mutant mice entered more often and spent more time in the former target (P < 0.05). These data reflect a higher ability of the mutant to remember accurately the location of the platform.
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Altogether, our study showed that spatial learning and memory capabilities in the MWM were improved in Ndn-deficient mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Our analyses show an abnormal phenotype of mice deficient for Ndn. Lack of Ndn is correlated to an early postnatal lethality with a partial penetrance. The surviving mutants have been investigated for subtle modifications of the hypothalamus, which showed a reduced number of oxytocin- and LHRH-producing neurons. Finally, Ndn-deficient mice present a characteristic behavioral profile with higher skin scraping as well as an improvement in particular cognitive functions associated with spatial learning and memory capability. This is the first report of hypothalamic or behavioral alterations in a mouse model deficient for Ndn. Such a model is of great interest with regard to the features of PWS.
An abrogation of the mouse Ndn gene has been previously reported by two different groups. Tsai et al. (22) observed no abnormal phenotype in Ndn-deficient mice, whereas Gérard et al. (21) observed postnatal respiratory distress leading to lethality in the first 30 h after birth. The penetrance of this phenotype was dependent on the genetic background. A high penetrance was observed in offspring of C57BL/6 mothers, in which males were more affected than females (95 versus 45%). The discrepancy between both reports could be attributed to differences between the mouse strains used for embryonic stem cell generation and breeding or to an unexpected contribution of the targeting construct to the phenotype (8). Here we report a third model of Ndn deficiency. We observed an early postnatal lethality on the 129Sv–C57Bl6/J genetic background. The penetrance of this phenotype varied according to the content of the C57Bl6/J genetic background, being increased in the N2 generation. Our data confirm the observations made by Gérard et al. (21) and support the hypothesis that modifier genes influence the phenotype of Ndn deficiency in the different mouse strains. However, we found a lower penetrance of lethality in the absence of sex-linked difference. These discrepancies might result from a divergence in the 129 and C57Bl/6 substrains used in both laboratories. Such a divergence has been suggested by Simpson et al. (35). Therefore, the lack of Ndn expression results in respiratory distress and neonatal lethality that resemble the respiratory problems often observed in PWS (3) and the failure to thrive of PWS patients (2).
Considering the relatively ‘mild phenotype’ associated with PWS, specifically the absence of obvious structural abnormalities in the brain, we did not expect gross defects in the absence of Ndn in mice. Thus, a detailed phenotypic analysis in search for more subtle changes was undertaken in order to define the biological role of the protein. In situ hybridization analysis showed an expression of Ndn in subpopulations of hypothalamic neurons. Quantification of hypothalamic oxytocin- and LHRH-producing neurons revealed a reduction of 29 and 25% of the populations analyzed, respectively. These data appear interesting with regard to the symptoms of PWS; obstetric problems and insatiable hunger have been suggested to be due to a lack of oxytocin (4) and hypogonadism to LHRH deficiency. A highly significant decrease in the number of oxytocin-expressing neurons (42%) was found in all five patients with PWS studied (36). LHRH-expressing neurons were not investigated in these patients. Preliminary data reveal undersized testes in Ndn-deficient males, which might be a consequence of reduction of LHRH neurons.
Specific behavioral and cognitive profiles represent important criteria of PWS (2,30,31,37). A general behavioral study was performed, blind to the genotype. These experiments demonstrated that Ndn deficiency does not affect behavioral responses related to motor coordination, exploratory activity, anxiety or stress. However, skin scraping activity in the open field was significantly elevated in mutants. Given that skin picking resulting from incessant scraping represents one of the main criteria described in PWS patients (32,38), this observation represents an interesting and important parallel.
The use of the MWM revealed that the abrogation of Ndn improves spatial learning and memory whereas it does not modify other behavioral responses affecting performance in this task. In order to understand how the inactivation of one gene may result in an improvement in learning capacities, one should keep in mind that the hidden platform version of MWM tests exclusively spatial learning and memory abilities (39). Consequently, better performance in this task does not test for a general improvement of cognitive function. Human genetic disorders, for example autism, Williams syndrome or PWS, are no longer considered as global disabilities, but as specific cognitive profiles with particular strengths and weaknesses. Consistent with these ideas, one of the strengths in PWS is visual–spatial integration and visual memory including an unusual skill to perform jigsaw puzzles (30,31,33,37).
An improvement in learning capacities due to the genetic manipulation of mice have so far been reported in few cases (40). Interestingly, enhanced performance in the MWM has been observed in animals following administration of a peptide hormone (39) or in mice lacking the neurotransmitter receptor (40). The latter findings imply that a disregulation in neuropeptide housekeeping can have consequences for spatial learning and memory. Thus, it can be hypothesized that Ndn deficiency may improve cognitive functions, at least in the MWM, through changes in neuropeptide levels and/or neurotransmitter activity, which remain to be determined.
A final important consideration is whether Ndn-deficient mice reflect the role of NDN deficiency in human PWS. Our analysis revealed striking parallels between the effects of this mutation in mice and phenotypic manifestations in PWS patients. However, it is clear that PWS is a multigenic disease showing a large variability of clinical features. The model presented here may consequently be representative of only a subset of these features although we cannot exclude that our analysis missed other important manifestations. More extensive anatomical, physiological and behavioral investigations will be necessary to determine whether there are further parallels between Ndn deficiency and PWS. In any event, Ndn-deficient mice represent a good model to investigate particular aspects of the molecular and physiological basis of respiratory distress, hypothalamic function and cognitive capacity and will be useful to investigate the role of Necdin in the central nervous system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Gene targeting and generation of Ndn-deficient mice
A mouse Ndn cDNA clone was used to screen the 129/Sv genomic DNA library of bacteriophage artificial chromosomes (BACs) produced by Research Genetics (Huntsville, AL). The positive BAC 143C10 was subcloned in pBluescript-SK after XbaI and BamHI digestion. The targeting vector was designed to delete 1.2 kb from XbaI to SacII, which contains the promoter and two-thirds of the coding part of Ndn. The plasmid contained a neo-selectable marker flanked by loxP sites on each side and an HSV-TK– selectable marker at the end of the construct. Using gancyclovir in the culture medium, this latter marker allows the elimination of clones in which a random integration occurred.
Vector DNA (15 µg) was linearized with NotI and electroporated into CK35-ES (129Sv genetic background) cells as described previously (42). G418-resistant clones were screened by Southern blotting using a 5' flanking probe corresponding to the XbaI fragment (Fig. 1b). Three positive clones among 1000 were expanded and injected into blastocysts by standard procedure (42).
Chimeric males were then bred to wild-type C57Bl6/J females or to females expressing CRE on C57Bl6/J genetic background (a gift from K. Rajewsky, Cologne, Germany). Southern blot analyses were performed as previously described (17).
Western blot analysis
Proteins were isolated with Trizol reagent (Life Technologie Tech-Line, Cergy Pontoise, France) from total frozen brain. Aliquots of 35 µg proteins were run in a 10% SDS–polyacrylamide gel, blotted over with transfer buffer (25 mM Tris base, 192 mM glycine, 20% methanol) at 4°C.
Immunodetection was performed with a 1:1000 dilution of polyclonal antipeptide AC2 antibody in Tween-20 [Tween–phosphate-buffered saline (PBS)] for 1 h at room temperature. The filters were washed three times for 15 min in 1x PBS, 0.1% Tween-20 and then incubated with a 1:5000 dilution of anti-rabbit Ig–horseradish peroxidase secondary antibody (Amersham Pharmacia Biotech, Orsay, France) in Tween–PBS for 1 h at room temperature.
Two polyclonal antibodies directed against the same synthetic peptides (C2 and N1) as designed by Aizawa et al. (28) were produced by Neosystem (Strasbourg, France). A chemiluminescence kit (ECL; Amersham) was used for visualization.
Northern and RT–PCR analysis
RNAs and cDNAs were prepared as previously described (18). Necdin PCR was performed using Nec3 (5'-TCTGGAGCAGGCCAGAGCTC-3') and Nec4 (5'-TGCTAAGTGCCTACACTGAG-3') primers and a 562 bp fragment was amplified. PCR conditions were 95°C for 2 min followed by 30 cycles of 95°C for 30 s, 50°C for 30 s and 72°C for 40 s. Neo PCR was performed using primers Neo1 (5'-TTTGTCAAGAACGACCTGTC-3') and Neo2 (5'-CGATACCGTAAAGCACGAGG-3') and a 598 bp fragment was amplified. PCR conditions were 95°C for 2 min followed by 30 cycles of 95°C for 45 s, 56°C for 45 s and 72°C for 45 s. Magel2 PCR was performed as described by Boccaccio et al. (17).
Snrpn PCR was performed using primers Snrpn1 (5'-GAGTAGCAAGATGCTGCAGC-3') and Snrpn2 (5'-GCCTCCCAACTCCTCTGACAG-3') and a 392 bp fragment was amplified. PCR conditions were 95°C for 2 min followed by 35 cycles of 95°C for 20 s, 55°C for 30 s and 72°C for 1 min.
Zfp127 PCR was performed using primers Zfp1 (5'-GTTCTTCCTTCTCTGATGAC-3') and Zfp2 (5'-CACAAGTTAACAAGTGCAC-3') and a 558 bp fragment was amplified. PCR conditions were 95°C for 2 min followed by 45 cycles of 95°C for 20 s, 49°C for 30 s and 72°C for 30 s.
In situ hybridization
Knockout (+m/–p) and wild-type mice were perfused intracardially using 4% paraformaldehyde in PBS. Brain was removed, frozen and stored at –80°C until use. Serial sagittal sections (14 µm) were obtained using a Microm cryostat and then collected onto silanized slides. Digoxigenin-labeled cRNA probes, specific for the 3'-UTR (from nucleotide 1174 after the ATG to nucleotide 1466) of Ndn, were obtained by in vitro transcription, Ndn mRNAs were detected as previously described (18,43).
Immunolabeling and counting LHRH- and oxytocin-producing neurons
The general protocol was described by Yoshida et al. (44). LR1 anti-LHRH polyclonal antibodies were diluted 1:30 000 and polyclonal anti-oxytoxin antibodies were diluted 1:10 000.
LHRH neurons were counted in knockout (+m/–p) and wild-type mice using 70 µm sagittal vibratome serial sections exploring 1.5 mm span containing the MPOA (Fig. 5). Oxytocin neurons were counted using 50 µm sagittal vibratome serial sections exploring 1 mm span beginning at the anterior part of the PVN and including supraoptic and accessory magnocellular nuclei located in lateral hypothalamus. Statistical analysis (Mann–Whitney non-parametric U-test) was performed using GB-STAT program 5.0.4 (Dynamic Microsystems, Silver Spring, MD).
Colocalization of LHRH protein and Ndn mRNAs in hypothalamic neurons
Double labeling experiments were performed using 50 µm vibratome slices. Floating sections were first subjected to in situ hybridization using our general protocol (see below). After the post-hybridization rinses, sections were incubated overnight at 4°C with LR1 anti-LHRH antibodies. After a rinse, sections were incubated with goat anti-digoxigenin antibodies coupled to alkaline phosphatase according to the supplier’s recommendations (Boehringer/Interchim/Roche, Meylan, France). Visualization of enzyme was performed using NBT and BCIP for 6 h. The sections were then incubated with anti-rabbit IgG conjugated to FITC, mounted in Mowiol and then placed on coverslips. Sections were viewed using a Leica DMR microscope. Digitized fluorescent or brightfield pictures were taken using a Princeton CoolSNAP camera (Roper Scientific, Trenton, NJ) and IPLab program 3.5 (Scananalytics, Fairfax, VA) running in a G4 Macintosh.
Spontaneous locomotor activity
Daytime activity was measured in a circular alley (outer diameter 20 cm; inner diameter 10 cm) equipped with eight photocells spaced evenly around the periphery. The number of interruptions of the photocell beams per 60 min was recorded automatically. The animals were placed in the apparatus at 10:00 h and were retrieved at 11:00 h. The sampling time was 5 min.
Elevated plus maze
The apparatus was made of four black metallic arms, two open arms (29 x 8 cm) and two enclosed arms (29 x 8 x 17 cm) that formed a cross shape with two arms opposite to each other. The maze was elevated 55 cm above the floor and placed in a dimly illuminated room (20 lux). Mice were placed individually on the central platform and allowed to explore the apparatus for 5 min. Anxiety was assessed by comparing the time spent in the open versus the enclosed arms. Furthermore, as an index of anxiety we used the ratio of (time spent in the open arms):(time spent in the open + closed arms). The latency for the beginning of exploration was also recorded.
Open field
The apparatus consisted of a white wooden box (0.8 x 0.8 x 0.5 m), the floor of which was divided into 25 squares. The arena was well illuminated (130 lux). Mice were placed on the central square and allowed to explore for 20 min. The sampling time was 5 min. Horizontal activity was the sum of the number of outer squares (those adjacent to the walls) crossed (outer locomotion) and of the number of inner squares crossed (inner locomotion). Horizontal activity and vertical activity (number of rearings) were computed as total activity. The latency to quit the first central square and the numbers of defecations, urinations, groomings and scrapings were also registered.
MWM
Spatial learning capacities were measured in an MWM (46). The apparatus consisted of a circular swimming pool built of gray plastic (180 cm diameter x 60 cm height), which was filled with water at room temperature and made opaque by the addition of milk. Four points on the rim of the pool divided the surface of the pool into four equal quadrants. During the test, the mice could escape onto a transparent perspex platform (10 x 10 cm) located in the middle of one of the quadrants. Starting points were chosen randomly in one of the three remaining quadrants. Spatial cues were placed in the room and were not moved during the experiment. The behavior of each animal was monitored and recorded by a videotrack system (View point, Lyon, France). Phase 1 involved no platform. During this habituation phase, animals were given one trial of 60 s per day over 3 days. Phase 2 involved a hidden platform. The mice were trained daily, for four consecutive trials of 90 s during 9 days, to escape onto a platform hidden just below the surface of the liquid (1.5 cm). The platform was kept in a constant position for the duration of this phase. The first trial was initiated by putting the animal on the platform for 30 s. If the animal jumped from the platform, it was replaced on the platform until it remained there for 30 s. The animal was then placed in one of the three quadrants. The trial ended when the mouse had climbed onto the platform where it was left for 30 s. If the animal did not find the platform in 90 s, it was placed there for 30 s. After this period of time, a new trial began. Phase 3 (probe trial). On the 7th day, a probe trial was performed. The platform was removed and animals were allowed to explore for 60 s. The number of entries in the platform zone and the time spent in the quadrant were recorded. Data were compared using Student’s t-test and, when necessary with an analysis of variance.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Supplementary material relating to this paper is available at http://www.hmg.oupjournals.org .
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ACKNOWLEDGEMENTS |
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We thank Marc Lalande, Christo Goridis, Willy Mayo for helpful discussions and support, Roger Keynes for critical reading of the manuscript; Nathalie Roëckel, Saïd Ech-Chadi and Elodie Drapeau for technical assistance; Charles Babinet and Chantal Kress (Pasteur Institute, France) for the gift of the CK35-ES cells; Robert Benoit (Montreal General Hospital, Montreal, France) for the gift of LR1 antibody; Gérard Tramu (Bordeaux, France) for the gift of oxytocin antibody; Robert Jeffard (Bordeaux, France) for the use of circular alleys material. This work was supported by grants from the Association Française contre les Myopathies (AFM), the Association pour la Recherche sur le Cancer (ARC), INSERM and Comité Mixte Inter-Universitaire Franco-Marocain.
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+ To whom correspondence should be addressed. Tel: +33 4 9178 6894; Fax: +33 4 9180 4319; Email: muscatel@ibdm.univ-mrs.fr
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