3 % N O S (V e c to r L aborato ries, B urlingam e, U S A ) 0.3 % T r ito n X -1 0 0 (B D H )
0.02 % N a N ^ (Sigma)
Chapter two M aterials and methods
2.2.2.
Drugs
M o rp h in e sulphate and cocaine H C l were purchased fro m Sigma. T h e y w ere dissolved in sterile saline (S te ri-A m p ; Steripak, R u n co rn , U K ) and adm inistered intraperitoneaU y (IP) using 0.5 m l M ic ro -F in e U -1 0 0 in s u lin syringes (B ecton D ic k in s o n , Le P o n t de C laix, France).
2.3.
Mice
A ll the experim ents described in this thesis used m ice. T h e m ice were housed in B io lo g ica l Services, U G L , L o n d o n , in cages co n ta in in g betw een 1 and 5 animals. E n v iro n m e n ta l c o n d itio n s w ere m a intained at 21
°C
and 50 % h u m id ity , w ith tap w a te r and fo o d (H arlan T e kla d T R M R a t/M o u s e D ie t; H a rla n , Bicester, U K )ad libitum.
L ig h ts were program m ed o n a 12:12 h cycle (lights o n at 8.00 am).2.2.1. Neurokinin-1 receptor knockout mice
T h e N K l re ce p to r k n o c k o u t (N K F ^ ) m ice used were derived fro m those described by D e Felipe
et al.
(1998). T h e k n o c k o u t Hne was created by targeted d is ru p tio n o f the gene en co d in g the N K l receptor, as illu stra te d in Figure 2.1. U sin g h o m o lo g o u s re c o m b in a tio n in e m b ry o n ic stem cells, a 1 2 9 /sv X C 5 7 B L /6 m ouse Hne was generated, in w h ic h exon 1 o f the gene was d isru p te d by in s e rtio n o f a cassette at a uniq u eSttA
site. T h e cassette consisted o f an in te rn a l rib o so m e entry site (1RES), thelacZ
co d in g sequence and a n e o m ycin resistance gene expressed fro m its o w n p ro m o te r.E x c e p t w here indicated, the N K 1 '^ ‘ m ice used in the experim ents described in this thesis were derive d fro m hom ozygous m u ta n t 1 2 9 /s v X C 5 7 B L /6 m ice crossed once o n to the M F l b a ckg ro u n d (H arlan; M ig a u d
et al
1998). T h is crossing procedure a llow ed a rapid d ilu tio n o f the 1 2 9 /s v c o m p o n e n t o f the m u ta n t m ice, since substrains o f this backgroun d p e rfo rm p o o rly in a n u m b e r o f b ehavio ural tests, in c lu d in g c o n d itio n e d place preference (CPP) and the M o rris w a te r maze, possibly due to th e ir h ig h anxiety levels (Baloghet al
1999; C raw leyet al
1997; D o cksta d e r & van der K o o y 2001; G e rla i 1996; H o m a n icset al
1999; M in e r 1997; O w e net al
1997; V o ik a ret al
2001).M ic e w ere b re d fro m hom ozygou s o r heterozygous breeding pairs. T h e m ice w ith in a ho m o zyg o u s b reeding p a ir were always progeny o f heterozygous m a tin g , to ensure that
Chapter two M aterials and methods
m u ta n t and c o n tro l m ice w ere o f sim ila r genetic backgroun d. M ic e w ere weaned at a p p ro xim a te ly three weeks o f age.
2.3.1.1.
Verification of genotype
T h e genotype o f aU m ice used in experim ents in v o lv in g N K T ^ ' m ice and w ü d type con tro ls was d e te rm in e d o r v e rifie d b y p e rfo rm in g the polym erase chain re a c tio n (PCR) o n D N A extracted fro m taü tip samples, as described in p ro to c o l 2.3.1.1 (see A p p e n d ix ). T h is p ro ce d u re uses three P C R prim ers: N K l - F and N K l - R am pHfy a 350-base section o f exon 1 o f the gene encodin g the N K l receptor, and N e o F and N K l - R am pHfy a 260-base section o f the N K l re ce p to r gene in c lu d in g the inserted cassette. R e s o lu tio n o f these P C R p ro d u c ts o n a 2 % agarose gel gives a single band at 260 bases fo r N K l ’^' m ice, bands at 260 and 350 bases fo r heterozygous m ice, and a single 350-base band fo r w ü d type mice.
2.3.2. Culling
M ic e w ere cuUed using COg asphyxiation, w ith the e xce p tio n o f those anim als u n dergo ing p e rfu s io n (see belo w ). M ic e w ere placed in a C O2 cham ber in to w h ic h a slow , steady flo w o f gas in to the cham ber was released, aUowing C O2 concentratio ns to rise slow ly. W h e n th e m ice had stopped breathing , death was v e rifie d b y physical b re a kin g o f the neck, e xsangu ination o r re m o va l o f the brain.
Chapter two Materials and methods
1RES B G al MC1 Neo
MC1 TK ' \ E x o n t '