Marie-Pierre Moisan

Behavioral and neuroendocrine reactivity to stress in the WKHA/WKY inbred rat strains: a multifactorial and genetic analysis

Genetic factors have been shown to influence the nature and the intensity of the stress responses. In order to understand better the genetic mechanisms involved, we have studied the behavioral and neuroendocrine responses to novel environments in the WKHA/WKY inbred strains and we have investigated the genetic relationships between these traits in a segregating F2 intercross. The animals were submitted to behavioral tests known to provide both indices of activity and fear (activity cages, open field and elevated plus-maze). The plasma levels of prolactin, ACTH, corticosterone, glucose and renin activity were determined after a 10-min exposure to novelty. Our results showed that WKHA rats, compared to WKYs, were more active in a familiar as well as in novel environments. They exhibited also less anxiety-related behaviors and lower neuroendocrine responses. A principal component analysis performed on the behavioral F2 results defined three independent factors: general activity, anxiety and defecation, none of them being correlated with the neuroendocrine measures. Thus this study suggests that these different responses to stress are independent components that may have distinct molecular bases.

GR 127935 reduces basal locomotor activity and prevents RU 24969-, but not D-amphetamine-induced hyperlocomotion, in the Wistar-Kyoto Hyperactive (WKHA) rat

Abstract The hyperlocomotor effect of the serotonin (5-HT)1A,B receptor agonist 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) has been repeatedly reported. However, 5-HT1A receptors, 5-HT1Bu2008receptors (or both) have been claimed to mediate this effect of RU 24969. These contradictory data possibly arise from protocol differences, especially those related to animal species, drugs, and activity assessment. Herein, the influence of a pretreatment with the selective 5-HT1B,D receptor antagonist N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiozol-3-yl)-biphenyl-4-carboxamide (GR 127935; 1, 3.3 and 10u2008mg/kg IP) on the hyperlocomotor effect of a 5u2008mg/kg (IP) dose of RU 24969 was studied in Wistar-Kyoto Hyperactive (WKHA) rats. In a first series of experiments, it was confirmed that RU 24969 (2.5 and 5u2008mg/kg), administered 10u2008min after the onset of activity recordings, increases locomotion dose-dependently (cage crossings). In a second series of experiments, administration of GR 127935 10u2008min after the onset of activity recordings promoted a dose-dependent decrease in basal activity (and rearings) and prevented (3.3 and 10u2008mg/kg) RU 24969-elicited locomotor activity. On the other hand, GR 127935 was ineffective against RU 24969-induced inhibition of rearings. Lastly, it was observed that 3.3u2008mg/kg GR 127935 did not affect the number of cage crossings and rearings displayed by rats administered 1.5u2008mg/kg D-amphetamine. This study shows that 5-HT1B receptors play a major role in the hyperlocomotor effect of RU 24969, at least under our experimental setting. Whether these receptors also play a tonic role in the high locomotor activity displayed by WKHA rats remains to be determined.

Rat strain differences in peripheral and central serotonin transporter protein expression and function

Female Fischer 344 (F344) rats have been shown to display increased serotonin transporter (5‐HTT) gene expression in the dorsal raphe, compared to female Lewis (LEW) rats. Herein, we explored, by means of synaptosomal preparations and in vivo microdialysis, whether central, but also peripheral, 5‐HTT protein expression/function differ between strains. Midbrain and hippocampal [3H]paroxetine binding at the 5‐HTT and hippocampal [3H]serotonin (5‐HT) reuptake were increased in male and female F344 rats, compared to their LEW counterparts, these strain differences being observed both in rats of commercial origin and in homebred rats. Moreover, in homebred rats, it was found that these strain differences extended to blood platelet 5‐HTT protein expression and function. Saturation studies of midbrain and hippocampal [3H]paroxetine binding at the 5‐HTT, and hippocampal and blood platelet [3H]5‐HT reuptake, also revealed significant strain differences in Bmax and Vmax values. Although F344 and LEW rats differ in the activity of the hypothalamo‐pituitary‐adrenal (HPA) axis, manipulations of that axis revealed that the strain differences in hippocampal [3H]paroxetine binding at 5‐HTTs and [3H]5‐HT reuptake were not accounted for by corticosteroids. Hippocampal extracellular 5‐HT levels were reduced in F344 rats, compared to LEW rats, with the relative, but not the absolute, increase in extracellular 5‐HT elicited by the local administration of citalopram being larger in F344 rats. Because the aforementioned strain differences did not lie in the coding sequences of the 5‐HTT gene, our results open the promising hypothesis that F344 and LEW strains model functional polymorphisms in the promoter region of the human 5‐HTT gene.

Functional Implication of an Arg307Gly Substitution in Corticosteroid-Binding Globulin, a Candidate Gene for a Quantitative Trait Locus Associated With Cortisol Variability and Obesity in Pig

We previously reported that corticosteroid-binding globulin gene (Cbg) may be the causal gene of a quantitative trait locus associated with cortisol levels, fat deposition, and muscle content in a pig intercross. Sequence analysis of parental animals allowed us to identify four amino-acid substitutions. Here we have examined if any of these single amino acid substitutions could be responsible for the difference in CBG binding and affinity for cortisol between the parental breeds, using in vitro assays of Cbg variants after transfection of mammalian cells. Additionally, the Cbg coding region was analyzed in samples from a synthetic pig line to study association between polymorphism and CBG biochemical properties, carcass composition, and meat quality. Both in vitro transfection assays and the association studies suggest a role of the Arg307Gly mutation in increasing CBG capacity (by >70%) and decreasing CBG affinity for cortisol (by 30%). The Ile265Val substitution may also have an effect on decreasing CBG affinity for cortisol by 25%. The mutations Ser15Ile and Thr257Met do not seem to have an effect on CBG parameters. The Arg307Gly substitution was the only mutation associated with a parameter of meat quality and no mutation was linked to carcass composition.

Marker-assisted selection of a neuro-behavioural trait related to behavioural inhibition in the SHR strain, an animal model of ADHD

The search for the molecular bases of neuro‐behavioural traits in Spontaneously Hypertensive Rats (SHR), an animal model of Attention Deficit Hyperactivity Disorder (ADHD), led to the discovery of two quantitative trait loci related to the locomotor activity in the centre of the open field. In the present study, rats from an F2 intercross between the SHR and Lewis strains were selected with markers on the basis of their genotype at these two loci. We obtained a ‘high line’ in which rats have the alleles increasing the trait, and a ‘low line’ with the lowering alleles. In activity cages with a dim light, the low line was more active than the high line. The reverse was found in the open field, and the inhibition of locomotor activity in the low line (as compared to the high line) was directly related to the aversiveness of the situation (larger in the centre than in the periphery, and in high light than in low light), and was more intense in males than in females. This inhibition is not attributable to a classical ‘anxiety’ factor as measured in the elevated plus maze, in which the open arms behaviours were not different between the lines. The high line also showed a deficit in prepulse inhibition of the acoustic startle reflex. The present data show that the two loci previously described in a SHRu2003×u2003Lewis intercross as related to the activity in the centre of the open field are indeed involved in a behavioural inhibition trait. The marker‐based selected lines described here are unique tools for the study of the neurobiological bases of this trait and the molecular foundations of its variability of genetic origin.

QTL mapping for traits associated with stress neuroendocrine reactivity in rats.

In the present study we searched for quantitative trait loci (QTLs) that affect neuroendocrine stress responses in a 20-min restraint stress paradigm using Brown–Norway (BN) and Wistar–Kyoto–Hyperactive (WKHA) rats. These strains differed in their hypothalamic–pituitary–adrenal axis (plasma ACTH and corticosterone levels, thymus, and adrenal weights) and in their renin–angiotensin–aldosterone system reactivity (plasma renin activity, aldosterone concentration). We performed a whole-genome scan on a F2 progeny derived from a WKHA × BN intercross, which led to the identification of several QTLs linked to plasma renin activity (Sr6, Sr8, Sr11, andSr12 on chromosomes RNO2, 3, 19, and 8, respectively), plasma aldosterone concentration (Sr7 and Sr9 on RNO2 and 5, respectively), and thymus weight (Sr10, Sr13, andSrl4 on RNO5, 10, and 16, respectively). The type 1b angiotensin II receptor gene (Agtrlb) maps within the confidence intervals of QTLs on RNO2 linked to plasma renin activity (Sr6, highly significant; LOD = 5.0) and to plasma aldosterone level (Sr7, suggestive; LOD = 2.0). In vitro studies of angiotensin II–induced release of aldosterone by adrenal glomerulosa cells revealed a lower receptor potency (log EC50 = −8.16 ± 0.11 M) and efficiency (Emax = 453.3 ± 25.9 pg/3 × 104 cells/24 h) in BN than in WKHA (log EC50 = −10.66 ± 0.18 M; Emax = 573.1 ± 15.3 pg/3 × 104 cells/24 h). Moreover, differences in Agtr1b mRNA abundance and sequence reinforce the putative role of the Agtr1b gene in the differential plasma renin stress reactivity between the two rat strains.

Further dissection of a genomic locus associated with behavioral activity in the Wistar–Kyoto hyperactive rat, an animal model of hyperkinesis

Molecular genetic studies of attention-deficit hyperactivity disorder (ADHD) are a major focus of current research since this syndrome has been shown to be highly heritable.1 Our approach has been to search for quantitative trait loci (QTL) in a genetic animal model of hyperkinesis, the Wistar–Kyoto hyperactive (WKHA) rat, by a whole-genome scan analysis. In a previous article, we reported the detection of a major QTL associated with behavioral activity in an F2 cross between WKHA and Wistar–Kyoto (WKY) rat strains.2 Here, we extend our analysis of this cross by adding new genetic markers, now defining a 10u2009cM interval on rat chromosome 8 associated with ambulatory and exploratory activities. Then we present a replication of this QTL detection, at least for exploratory activity, by a new genetic mapping analysis of an activity QTL in an F2 cross between the WKHA and Brown Norway (BN) rat strains. Overall, the results provide compelling evidence for the presence of gene(s) influencing activity at this locus. The QTL interval has been refined such that the human orthologous region could be defined and tested in human populations for association with ADHD. Ultimately, the improved dissection of this genomic locus should allow the identification of the causal genes.

Integrated genetic mapping of 64 rat microsatellite markers from different sources

The rat (Rattus norvegicus) is a major animal model for biomedical research (Gill et al. 1989). In particular, the rat is a model of choice in neurobiology, behavioral biology, and endocrinology, in great part because of its larger size (compared with the mouse) that facilitates experimental interventions. In contrast to its central role in the study of physiology, the use of the rat as a genetic animal model has long been limited by the lack of a rat genetic map. The recent publication of rat genetic maps (Serikawa et al. 1992; Jacob et al. 1995; Pravanec et al. 1996) has opened new perspectives, and

Linkage mapping of α3, α5, and β4 neuronal nicotinic acetylcholine receptors to rat Chromosome 8

GGAGAGCGCTTGCTGAGCTCC-3 [4]; P4hb, (upstream) 5GTCTTCATGTCCAGCTACTTG-3 and (downstream) 5GGCTGGTCGGTAGTCCTGG-3 [4]. Allele detection: An RFLV for Grp58 was identified with Tagl, where fragments of 3.0 kb in C57BL/6J and 3.6 kb in M. spretus were used for scoring. An RFLV for Erp72 was identified with BglIJ, which generated fragments of 5.2 and 6.8 kb for C57BL/6J and 5.2 and 5.8 kb for M. spretus. An RFLV for P4hb was identified with PvuII, which produced a fragment of 3.5 kb for C57BL/ 6J and 3.8 kb for M. spretus. Previously identified homologs: The mouse PDI gene (P4hb) was mapped previously to the distal region of Chr II with a [(C57BL/ 6J x Mus spretus)F 1 x C57BL/6J] interspecific cross [8]. The human PDI gene has been mapped by somatic cell hybrids to Chr 17q25 [5]. The human Grp58 is located on Chr 15g15 [6]. To date, no homolog for mouse Erp72 has been mapped in humans. No known mutations are present in the regions where P4hb, Erp72, and Grp58 are located. Abbreviations: For discussion purposes, we designate the Grp58, gene product as Grp58 (glucose-regulated protein 58), although it has also been called PI-PLC, ERp57, ERp60, ERp61, and PDI-Q2. The Erp72 gene product is called ERp72 (endoplasmic reticulum protein 72). We designate the P4hb gene product as PDI (protein disulfide isomerase), although it has also been called ERp59. Discussion: The PDI family represents a group of thioredoxinrelated proteins that reside in the lumen of the ER [7]. Here we report the chromosomal position for three members of this family, Grp58, Erp72, and P4hb (Fig. 1). P4hb, mapped previously in an interspecific cross identical to the one used here, placed this locus about 12 cM distal to Pkca on Chr 11 [8]. Figure 1 shows that this map position is very similar to ours, placing P4hb about 11 cM from Pkca. The PDI family is characterized by the presence of thioredoxin-like domains containing the motif CGHC; this motif contains the redox-active cysteine residues involved in the disulfide isomerase activity of PDI [7,9]. The PDI family members are soluble proteins that are retained within the ER by the C-terminal retention signals -KDEL, -KEEL, and -QEDL for PDI, ERp72, and Grp58 respectively [10]. PDI is the best characterized protein of this gene family and is well known for its function as a proteinfolding enzyme that catalyzes disulfide bond isomerization [11]. PDI has the capacity to bind a wide variety of peptides at a domain separate from its CGHC active site, and this domain may function to target PDI to newly synthesized polypeptides as they are translocated into the ER [11]. In addition, PDI is part of complex enzyme systems operating in the ER: PDI is a subunit of the enzyme prolyl-4-hydroxylase, catalyzing the formation of hydroxyprolyl residues in nascent collagen-like polypeptides; PDI is also a subunit of the microsomal triacylglycerol transfer protein, functioning in the formation of nascent, triglyceride-rich lipoproteins [7]. While ERp72 and Grp58 also contain thioredoxin-like domains, they do not display PDI activity in vitro [12]. Nevertheless, it has been shown that ERp72, but not Grp58, can replace the growth-essential PDI function in yeast [12]. In addition, ERp72 is involved in ER-specific protein degradation [13], and Grp58 has recently been implicated as a glycoprotein-specific molecular chaperone [14].

Assignment of the gene encoding the serotonin 5HT1B receptor to rat Chromosome 8q31 by fluorescence in situ hybridization

Species:Mouse Locus name: methionine synthase or 5-methyltetrahydrofolatehomocysteine methyltransferase Locus symbol:Mtr Map position: proximal–D13Mit1–1.06 cM ± 1.06 SE– Mtr, D13Bir4, D13Bir6–1.06 ± 1.06–D13Abb1e–2.13 ± 1.49–D13Bir7–distal Method of mapping:Mtr was localized by RFLP analysis of 96 animals from an interspecific backcross panel ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei) provided by The Jackson Laboratory, Bar Harbor, Me. (BSS panel) [1]. Database deposit information: The data are available from the Mouse Genome Database, accession number MGD-JNUM-39061. Molecular reagents:A 1095-bp mouse cDNA was obtained by reverse transcription/PCR of mouse liver RNA, with degenerate oligonucleotides based on regions of homology within the methionine synthase sequences of lower organisms. The two primers (D1730 and D1733), as described by Leclerc et al. [2], were successful in amplifying both human and mouse cDNAs. The PCR products from both species were subcloned and sequenced; they showed 89% identity. The mouse cDNA was labeled by random priming and hybridized to Southern blots of EcoRI-digested mouse genomic DNA. Allele detection:Allele detection was performed by RFLP analysis of an EcoRI polymorphism. The C57BL/6J strain has alleles of approximately 13 kb, while theMus spretusstrain has alleles of approximately 9 kb and 4 kb. A constant band of approximately 0.5 kb was seen in both strains. Previously identified homologs: Human MTR has been mapped to chromosomal band 1q43 by fluorescence in situ hybridization [2–4]. Discussion: Methionine synthase (EC 2.1.1.13, 5-methyltetrahydrofolate-homocysteine methyltransferase) catalyzes homocysteine remethylation to methionine, with 5-methyltetrahydrofolate as the methyl donor and methylcobalamin as a cofactor. Nutritional deficiencies and genetic defects in homocysteine metabolism result in varying degrees of hyperhomocysteinemia. Dramatic elevations in plasma and urinary homocysteine levels are associated with the inborn error of metabolism, homocystinuria. Consequent to the recent isolation of the human cDNA for methionine synthase [2–4], two groups of investigators have identified mutations in methionine synthase in homocystinuric patients [2, 5]. Mild elevations in plasma homocysteine are thought to be a risk factor for both vascular disease and neural tube defects [6–8]. A genetic variant in methylenetetrahydrofolate reductase (MTHFR), the enzyme that synthesizes 5-methyltetrahydrofolate for the methioninesynthase reaction, is the most common genetic determinant of hyperhomocysteinemia identified thus far [9]. Mild defects in the methionine synthase reaction are also potential candidates for hyperhomocysteinemia and the associated multifactorial diseases. A common variant has been reported for the human methionine synthase gene, but its physiologic consequences have not yet been determined [2, 4]. The mapping of the human MTR gene to 1q43 and of the mouse gene to proximal Chromosome (Chr) 13 is consistent with previous findings of human/mouse homologies between these 2 chromosomal regions; the human and mouse nidogen genes have been mapped to 1q43 and proximal Chr 13, respectively [10]. Several genes have already been implicated in neural tube defects in mice [11]. Studies involving the mouse methionine synthase gene will be useful in assessing the role of this important enzyme in the development of birth defects and/or vascular disease.