The targeting vector, derived from a 129/OLA cosmid encompassing the Rgs4 locus (RZPD Resource Center, Berlin, Germany), consisted of an 11-kb 5′ homology arm and a 1.12-kb 3′ homology arm separated by an Flp recognition target-flanked neo cassette itself preceded by a loxP/splice-acceptor site/promotorless lacZ cassette (Fig. ). The neo and lacZ cassettes replaced a 1.2-kb region containing low-complexity sequences with no similarity to the human locus, located 0.6 kb downstream of the Rgs4 poly(A) signal. Upstream (4.5 kb) of these two cassettes, a second loxP site and a third FRT site were introduced in the second intron of Rgs4, so that recombination by the Flp or Cre recombinase would interrupt the Rgs4 coding sequences before the RGS domain (Fig. ).
FIG. 1. Generation of Rgs4 deficient mice. (A) Targeting strategy. (Top) Wild-type Rgs4 allele with exons 1 to 5 (boxes, coding sequences in dark shade, non coding sequences in light shade). The RGS domain is encoded by exons 3, 4, and 5. Selected sites for EcoRI (more ...) Production of mice and genotyping.
A homologous recombinant clone of E14-1 embryonic stem (ES) cells (129/OLA) was obtained by electroporation and verified for the correct recombination event by Southern blotting using both internal and external probes (Fig. and not shown). Injection of the clone into C57Bl/6 blastocysts produced nine male chimeras, four of which transmitted the recombinant locus. Genotyping of Rgs4loxFRTlacZ/+
mice was performed by PCR on genomic tail DNA using primers which surround the loxP/FRT
insertion site in the second intron (SensInt2, 5′-GGTGAGGGTACACAATTGTAG-3′; antiEx 4, 5′GCTTTGAAAGCTGCCAGTCC-3′) and produce a 566-bp and a 693-bp band from the wild type (wt) and the targeted allele, respectively (Fig. ). Rgs4loxFRTlacZ/loxFRTlacZ
males were crossed with heterozygous PGK-Cre
) (on a B6D2 background), and the offspring was genotyped by PCR using two pairs of primers: one amplifying a 780-bp band from the Cre-recombined allele (SensInt2 [see above] and AntilacZ: 5′-GGCGCATCGTAACCGTGCAT-3′), the other amplifying a 519-bp band from the wild-type allele (SensEx4, 5′-CAACGAGTTCATCTCAGTGCCA-3′; AntiEx5, 5′-TGGGCTGTAACATGTTCCGG-3′) (Fig. ).
All strains of mice used in this work were bought at Janvier.
Embryos (the day of the vaginal plug was considered as embryonic day 0.5 [E0.5]) were stained with X-Gal (5-bromo-4-chloro-3-indolyl-β-d
-galactopyranoside) as described by Knittel et al. (25
) and for in situ hybridization combined or not combined with immunohistochemistry as described by Tiveron et al. (44
). Adult tissues were treated as described by Grillet et al. (17
). Golgi stains were done with the rapid GolgiStain kit (FD Neurotechnologies) according to the manufacturer's instructions.
In situ hybridization probes were pKS-mRgs4
), class III β-tubulin (kind gift of C. W. Ragsdale), and lacZ
, and antibodies were specific for Phox2a (44
), Phox2b (36
), or Islet1/2 (kind gift of T. Jessell).
General health assessment and gross neurological examination were performed, with slight modifications, as previously described (19
) on 11 wt and 12 Rgs4lacZ/lacZ
male littermates, 13 to 15 weeks old, born from matings of heterozygotes, themselves descendants of three backcrosses on C57BL/6 of the original (mixed 129/B6D2) Cre-deleted mouse. All experiments were carried out in accordance with the European Community Council Directive (24 November 1986) at the Institut Clinique de la Souris (Illkirch, France) except for the morphine withdrawal test, which was carried out in the authors' lab. A more comprehensive description of each protocol can be found at http://www-mci.u-strasbg.fr/service_4.html
Behavioral tests. (i) Rotating rod test.
After three 60-s habituation trials on a rotating rod (Bioseb, Chaville, France) (one trial at 0 rpm and 2 trials at 4 rpm), mice were subjected to three testing trials (separated by 5- to 10-min intervals) during which the rotation accelerated from 4 to 40 rpm in 5 min and the time from latency to falling off was measured.
(ii) String test.
In three consecutive trials (separated by 10-min intervals), the animals were suspended by their forepaws on a horizontally stretched wire, and the time they took to catch the wire with their hind paws was recorded.
(iii) Grip test.
In three consecutive trials, the animals were allowed to grasp a grid connected to an isometric dynamometer (Bioseb) and slowly moved backwards until they released it. The maximal strength developed was expressed in grams per gram.
(iv) Tail flick test.
The tail of the animal was placed under a heat source (a shutter-controlled lamp [Bioseb]) and heated three times at different sites at intervals of 1 min. The time taken by the animal to flick its tail was recorded (cutoff point at 25 s).
(v) Hot-plate test.
In two trials, the mice were placed on a hot plate adjusted to 52°C (Bioseb), and the latency to the first reaction (licking, moving the paws, or leaping) was recorded. The test was ended if the mouse did not react within 30 s. In morphine experiments, the second trial duration was increased to 180 s maximum.
(vi) Shock threshold test.
Mice were allowed to habituate for 30 s to a fear conditioning chamber. Behavioral responses to a 1-s foot shock (beginning at 0.05 mA and increased in 0.05-mA steps at 30-s intervals) were recorded. When both flinch (any detectable response) and vocalization were induced, shocks were increased in 0.1-mA steps (cutoff point at 1 mA) until a jump (defined as the two hind paws leaving the ground) was induced.
(vii) Open field test.
Mice were tested in an automated open arena (Panlab, Barcelona, Spain) divided into central and peripheral regions homogeneously illuminated at 150 lx. Mice placed at the periphery of the open field were allowed to freely explore the apparatus for 30 min. The distance traveled, the number of rearings, and the time spent in the central and peripheral regions were recorded.
(viii) Tail suspension test.
Mice were fitted in a tail suspension device (MED Associates Inc., St. Albans, Vermont). The latency to the first immobilization was determined, and the immobility time was monitored during a 6-min period in blocks of 2 min.
(ix) Auditory startle reflex reactivity and prepulse inhibition.
Acoustic startle reactivity and prepulse inhibition of startle were assessed using a standard startle chamber (SR-Lab startle response system; San Diego Instruments) and 10 different trials: acoustic startle pulse alone (120 dB), prepulse trials with 70-, 80-, 85- or 90-dB stimuli presented either alone or preceding the pulse, and one trial in which only the background noise (65 dB) was presented to measure the baseline movement. The amount of PPI in the prepulse plus pulse trials was expressed as a percentage of the startle response in the pulse alone trial.
(x) Y-maze spontaneous alternation.
Y-maze spontaneous alternation was assessed in a Y maze with three arms (40 by 9 by 16 cm) placed at 120° from each other and distinguishable by specific motifs on their walls. Mice were placed at the end of one arm and allowed to freely explore the apparatus for 5 min. Alternations were defined as successive entries into each of the three arms. Performance was defined as the ratio of actual alternations to possible alternations × 100.
(xi) Pavlovian fear conditioning.
Pavlovian fear conditioning was assessed in 18.5- by 18- by 21.5-cm operant chambers (Coulbourn Instruments, Allentown, PA) with aluminium side walls and Plexiglas rear and front walls. A loudspeaker and a bright light, inserted on opposite walls, provided the cues during conditioning and cue testing. The activity of animals was monitored by infrared cells in the ceiling and recorded using the Graphic State software (Coulbourn). After a 4 min acclimatation, a light/tone (10 kHz) (conditional stimulus) was presented for 20 s and coterminated by a mild (1 s, 0.4 mA) foot shock (unconditional stimulus). Another conditional stimulus-unconditional stimulus pairing was presented 2 min later, and mice were returned to their home cages 2 min later. Twenty-four hours after the conditioning session, mice were tested for context by placing them back into the operant chamber and allowing them to explore it for 6 min. Testing for the cue was done 5 h after testing for context: mice were placed in a new chamber (with different wall color, odor, and floor texture), allowed to habituate for 2 min, and presented with light/auditory cues for 2 min. This sequence was repeated once.
(xii) Morphine withdrawal.
Mice were injected intraperitoneally twice a day, 10 h apart, for 5 days with escalating doses of morphine (two injections of 20, 40, 60, 80, and 100 mg/kg of body weight on days 1, 2, 3, 4, and 5, respectively). On the sixth day, withdrawal was precipitated 2 h after one administration of morphine at 100 mg/kg, by subcutaneous injection of naloxone at 1 mg/kg. Withdrawal behaviors were monitored and scored over a period of 30 min according to Celerier et al. (4
For quantitative parameters showing normal distribution, data were analyzed using the unpaired Student t test or repeated measures of analysis of variance (ANOVA) with one between factor (genotype) and one within factor (time). Qualitative parameters (e.g., some clinical observations) were analyzed using the χ2 test. The level of significance was set at a P value of <0.05. Morphine analgesia and withdrawal scores were analyzed using two-way ANOVA with genotype and treatment as factors of variation. When appropriate, one-way ANOVA and Student-Newman-Keuls post hoc analysis were performed.