5. RESULTADOS
5.1 EL CONTEXTO: EL COLEGIO DIDASCÁLICO HERMANA JOSEFINA SERRANO
6.5.1.2.1. Surgery
O n e h u n d re d and e ig h ty -tw o m ice were used in these experim ents. M ic e received bilateral in je ctio n s in to the amygdala u n d e r stereotaxic surgery, according to the coordinates given in T able 6.2. In je c tio n s were o f 1.0 p i 1.00 p M SP-SAP
(n —
78), 1.00 p M SP(n =
28; Sigma) o r 1.00 p M SAP(n =
40; A d va n ce d T a rg e tin g Systems). T h irty -s ix m ice d id n o t undergo surgery and were used as naïve controls. T w o m ice died d u rin g surgery (one each fro m the SP-SAP and SP groups), and three m ice died a fte r surgery (tw o fro m the SP-SAP g ro u p and one fr o m the SAP group).6.5.1.2.2. Behavioural analysis
B e g in n in g 5 weeks a fte r surgery, each o f the fo u r groups o f m ice (SP-SAP; SP; SAP; naïve) was su b d ivid e d in to tw o fu rth e r groups. T h e n u m b e r o f m ice in each g ro u p is given in T able 6.3.
Chapter six Behavioural effects o f ablation o f neurones expressing the neurokinin-1 receptor
T a b le 6,3 N u m b e rs o f m ice used in m o rp h in e experim ents.
T re a tm e n t G ro u p I G roup II T o ta l SP-SAP 38 37 75 SP 15 12 27 SAP 21 18 39 Naïve 18 18 36 T o ta l 92 85 177
M ice in b o th groups w ere assessed fo r anxiety b e h a vio u r using the E P M (see above). T hereafter, the tw o groups w ere treated separately: G ro u p I m ice u n d e rw e n t m o rp h in e CPP and the lo c o m o to r response to saline, w h ils t G ro u p I I m ice u n d e rw e n t saline CPP and testing fo r the lo c o m o to r response to acute m o rp h in e . T h e o rd e r o f the tw o tests was swapped betw een the tw o groups to ensure th a t m o rp h in e in je c tio n s occurred d u rin g the fin a l beh a vio u ra l test. M ic e u n d e rw e n t surgery and w ere tested in groups o f 12 over a p e rio d o f a p p ro xim a te ly 18 m o n th s — each g ro u p containe d m ice fro m any o r aU o f the fo u r tre a tm e n t groups.
O n e day a fte r testing o n the E P M , m ice in G ro u p I u n d e rw e n t testing o f the lo c o m o to r response to an acute in je c tio n o f saline in the open fie ld (see section 2.5.4.1). M ice were g iven an in je c tio n o f sterile sahne (S te ri-A m p ; 10.0 ml.kg'^) and re tu rn e d to the hom e cage. T h ir ty m in u te s a fte r in je c tio n , lo c o m o to r a c tiv ity was m o n ito re d fo r 10 m in . B eginnin g 3 — 5 d a fte r lo c o m o to r assessment, they w ere then tested fo r CPP to m o rp h in e (3.0 mg.kg"^), given m a v o lu m e o f 6.67 ml.kg'^ (see section 2.5.3). T h irty -e ig h t animals dem onstrate d a preference fo r one o f the com p a rtm e n ts d u rin g p re c o n d itio n in g (13 fro m the SP-SAP g ro u p , 6 fro m the SP g roup, 13 fro m the SAP g ro u p and 6 fro m the naïve g ro u p ) and w ere excluded fro m the rem ainder o f the beh a vio u ra l experim ent.
B e g in n in g 1 d a fte r testing o n the E P M , G ro u p I I m ice u n d e rw e n t CPP w ith saline (6.67 m l.k g '\ see section 2.5.3). M ice w h ic h dem onstrate d a preference fo r one o f the tw o co m p a rtm e n ts d u rin g p re c o n d itio n in g (14 fro m the SP-SAP g ro u p , 3 fro m the SP g roup, 9
Chapter six Behavioural effects o f ablation o f neurones expressing the neurokinin-1 receptor
fro m the SAP g ro u p and 4 fro m the naïve g roup) were n o t re m o ve d fro m the experim ent, b u t assigned to receive the firs t in je c tio n o f saline in one o f the tw o com partm ents at random . T h e y u n d e rw e n t the entire CPP procedure b u t th e ir CPP scores w ere o m itte d fro m statistical analysis. T h re e to five days after the test session, the lo c o m o to r effects o f an acute in je c tio n o f m o rp h in e (10.0 mg.kg'^) were assessed in the o pen fie ld (see section 2.5.4.1). T h e m ice w ere given an in je c tio n o f m o rp h in e in a v o lu m e o f 10.0 m l.kg^ and re tu rn e d to the ho m e cage. T h ir ty m inutes a fte r in je c tio n , lo c o m o to r a c tiv ity was m o n ito re d fo r 10 m in.
6.5.1.2.3. Immunohistochemistry
A fte r the b e h a vio u ra l experim ents, m ice w ere perfused, and th e ir brains rem oved, post fixed, cryo p ro te c te d and sectioned (see section 2.4.1). IH C was carried o u t fo r the N K l receptor, N e u N and G F A P as described above.
6.5.1.2.4. Analysis
E x a m in a tio n o f im m unohistoch em icaU y-stained sections was carried o u t indepe n d e n tly by b o th experim enters w h o w ere b lin d e d to the results o f beh a vio u ra l testing. Results were com pared and agreed betw een the tw o observers.
T h e p o s itio n and e xte n t o f loss o f N K l receptor-expressing neurones in the amygdala was assessed in the brains o f m ice fro m the SP-SAP group. T h e c rite ria fo r in c lu sio n in the analysis w ere m o re strin g e n t than in the p re lim in a ry experim ents described above: animals were o m itte d fro m analysis unless they had a clear re d u c tio n o f N K l receptor im m u n o re a c tiv ity th ro u g h o u t the amygdala o n b o th sides o f the brain. A n im a ls w ith visib le necrosis o r b ila te ra l loss o f N K l receptor-expressing neurones in any o th e r brain region w ere also o m itte d . F o r the re m a in in g three groups o f m ice (SP, SAP and naïve), the in te g rity o f N K l re c e p to r im m u n o re a c tiv ity was checked th ro u g h o u t the brain. In aU groups, n o n -s p e c ific loss o f neurones was assessed in sections processed fo r N e u N IH C , w h ils t gliosis was sought in sections processed fo r G F A P .
F o rty -e ig h t m ice fr o m the SP-SAP gro u p w ere o m itte d fro m analysis o n the basis o f the above c rite ria (23 fro m G ro u p I and 25 fro m G ro u p II) . N o m ice w ere o m itte d fro m the SP o r SAP groups.
Chapter six Behavioural effects o f ablation o f neurones expressing the neurokinin-1 receptor
T h e tw o measures o f b e h a vio u r o n the E P M (tim e in open arms and n u m b e r o f entries in to o p e n arm s) w ere com pared across groups using 1-way A N O V A s w ith tre a tm e n t (SP- SAP, SP and SAP) as the betw een-subjects fa c to r (see section 2.6).
Post hoc
T u ke y com parisons w ere used fo llo w in g sig n ifica n t m ain effects. T w o m ice fro m the SP g ro u p were o m itte d : one d id n o t m o ve fro m the central zone o f the maze d u rin g the 5 m in re c o rd in g , and the o th e r ju m p e d fro m the maze soon a fte r the start o f the trial. F o r lo g is tic a l reasons, 12 m ice fro m the naïve g ro u p w ere n o t tested o n the E P M . T h e fin a l n um bers o f m ice in each g ro u p are given in T a b le 6.4.CPP scores fr o m anim als w ith o u t preference d u rin g p re c o n d itio n in g and w h ic h were n o t o m itte d o n the basis o f h is to lo g y were com pared using a 2 -way A N O V A w ith treatm ent and d ru g as betw een -su bject factors.
Post hoc
analysis was carried o u t using T u k e y com parisons (see section 2.6). D u e to considerable v a ria b ility in the data, CPP scores th a t fe ll m o re th a n 2 SDs fro m the g ro u p m ean w ere excluded fro m analysis (one fro m the m o rp h in e SP g ro u p and tw o fro m the m o rp h in e SAP group). A s tw o fu rth e r m ice fro m the m o rp h in e SAP g ro u p were given in je ctio n s in the w ro n g c o m p a rtm e n t o f the CPP apparam s d u rin g c o n d itio n in g , th e ir scores were also excluded fro m analysis. T h e fin a l num bers o f m ice in each g ro u p are given in T able 6.4.T h e measure o f lo c o m o to r a c tiv ity was analysed using a 2-w ay A N O V A w ith tre a tm e n t and d ru g as b etw een -su bject factors, fo llo w e d b y
post hoc
T u k e y com parisons (see section 2.6). T h e lo c o m o to r re c o rd in g e q u ip m e n t m a lfu n c tio n e d o n m any o f the trials. A s such, the n u m b e r o f m ic e in clu d e d in the fin a l analysis was lo w . D a ta fro m 3 m ice were also excluded fr o m analysis as they fe ll m o re than 2 SDs fro m the g ro u p m ean (one fro m the m o rp h in e naïve g ro u p , and tw o fro m the m o rp h in e SP-SAP g roup). T h e fin a l num bers o f m ice in each g ro u p are given in T able 6.4.Chapter six Behavioural effects o f ablation o f neurones expressing the neurokinin-1 receptor
T able 6.4 Final num bers o f m ice in analysis
T reatm ent E PM CPP L ocom otor assessm en t