PROTECCIONES PARA LA FUENTE DE ALIMENTACIÓN

T h e experim ents presented in this thesis use a w id e range o f d iffe re n t b ehavio ural techniques in the assessment o f the re la tio n sh ip o f the N K l re c e p to r to rew ard and le a rn in g and m e m o ry processes. H o w e v e r, the o n ly techniques th a t are used in m o re than one chapter o f this thesis are CPP and lo c o m o to r assessment, w h ic h are explained below .

2.5.1. General principles

T h e analysis o f a n im al b e h a vio u r is a useful way in w h ic h lin ks betw een neural processes and h ig h e r fu n c tio n s , such as rew ard and learning can be assessed. D u e to the co m p le x n a ture o f the c o n tro l o f b ehavio ur, care has to be taken to ensure th a t conclusion s draw n fr o m b e h a vio u ra l w o r k are n o t affected b y c o n fo u n d in g variables, such as stress and anxiety. Since elevated anxiety levels can im pede an a nim al’s b e h a vio u r, h a b itu a tio n to h a n d lin g and the test apparatus b e fo re the start o f the e xp e rim e n t is o fte n necessary to

Chapter two M aterials and methods

m in im is e the effects o f anxiety. T h e presence o f the e xperim ente r in the testing ro o m can also increase anxiety levels. T o a vo id this, b e h a vio u ra l experim ents w ere carried o u t in a dedicated ro o m , w ith as little external disturbance as possible. W h e re the nature o f the e xp e rim e n t allow ed, experim ents were videotap ed o r m o n ito re d using a PC , w h ile the e xp e rim e n te r w a ite d outside the b e h a vio u r ro o m .

In m o s t cases, each m ouse was o n ly used in one type o f b e h a vio u ra l e xperim ent. W here it was necessary to use a m ouse o n m o re than one occasion, a tim e p e rio d o f at least a week was in c lu d e d betw een tw o experim ents, and m ice w ere never used fo r a ne w expe rim e n t a fte r one in v o lv in g d ru g a d m in istra tio n . Because o f the adverse effects o f singular h ousing on b e h a v io u r (B ardo

et al.

1995), o n ly m ice housed in groups w ere used fo r b ehavio ural studies, and a lb in o anim als were avoided due to th e ir p o o r visu a l acuity (C raw ley

et al.

1997; O w e n

et al.

1997; Rhoades & H e n ry 1977). E x c e p t w here in dicated , b ehavio ural experim ents w ere carried o u t u n d e r flu o re sce n t s trip lig h tin g , at an in te n s ity o f 40 Ix. M ice w ere tra n sp o rte d in to the b e h a vio u ra l ro o m at least 24 h b e fo re the start o f b ehavio ural testing.

2.5.2. EthoVision

T h e b e h a vio u ra l m o n ito rin g softw are E th o V is io n 2.3 (N o ld u s In fo rm a tio n T e ch n o lo g y, W ageningen, T h e N etherland s) was used in m any o f the experim ents described in this thesis. I t converts analogue visu a l in fo rm a tio n fro m a v id e o camera (H ig h R e solution B / W C C D Camera M o d e l V C B -3 3 7 2 P ; Sanyo E le c tric C om pany, Osaka, Japan) to a dig ita l signal, w h ic h is used to m o n ito r the p o s itio n o f anim als in up to 8 e xp e rim e n ta l arenas sim ultaneously. In the ‘su b tra c tio n ’ m o d e used in the experim ents presented here, it com pares the im age received fro m the camera to a p re vio u sly collected reference image o f the e xp e rim e n ta l apparatus, consid e rin g the largest area o f discrepancy betw een these tw o images in each arena to be the an im a l u n d e r observation. I n all the experim ents described in this thesis, images w ere collected at a frequency o f 5 H z . F o llo w in g re co rd in g , a w id e v a rie ty o f param eters can be calculated fo r each anim al b e in g observed. These in clu d e the tim e spent in u ser-define d zones w ith in each experim enta l arena, the speed o f m o v e m e n t and n u m b e r o f rearings (characterised b y large reductions in the an im a l’s surface area).

Chapter two M aterials and methods

2.5.3. Conditioned piece preference

CPP is a beh a vio u ra l technique fo r assessing the re w a rd in g prop e rtie s o f a d ru g o r other stim ulus (see B a rd o & Bevins 2000). A typical CPP e xp e rim e n t consists o f the d iffe re n tia l p a irin g o f one o f tw o sets o f co n te xtu a l o r e n v iro n m e n ta l cues w ith the stim ulus o f interest. T h e c o n te x tu a l cues tend to d iffe r in term s o f w a ll c o lo u r o r p a tte rn , flo o r texture, sm ell, size o r shape, and the anim al is exposed to repeated pairings o f the stim ulus w ith one co n te xt, interspersed w ith exposures to the o th e r c o n te x t b u t w ith o u t the co n d itio n e d stim ulus. F o llo w in g c o n d itio n in g , the anim al is given a choice test, in w h ic h i t is given free access to b o th e n viro n m e n ts, and the a m o u n t o f tim e spent in each is m o n ito re d . A n increase in the a m o u n t o f tim e spent in the e n v iro n m e n t paired w ith the stim ulus o f in te re st is taken as evidence th a t the stim ulus was rew arding, whereas a decrease indicates th a t it was aversive. In such cases, the paradigm is o fte n refe rre d to as c o n d itio n e d place aversion (C PA).

CPP has been used to dem onstrate the re w a rd in g properties o f b o th natu ra l and drug stimuH. N a tu ra lly o c c u rrin g s tim u li th a t can b rin g a b o u t an increase in preference in rodents in clu d e fo o d , water, sweet fluids, in te ra c tio n w ith conspecifics, ejaculation, c o p u la tio n and n o v e l stimuH, a lthoug h these tend to be subject to changes in m o tiv a tio n a l state, such as h unge r fo r fo o d reward. A w id e va rie ty o f drugs can also induce preference. T h e abused drugs w h ic h have been show n to induce CPP in clu d e p sychostim ulan ts (e.g. cocaine and am phetam ine), opiates (e.g. m o rp h in e and h e ro in ), n ic o tin e , ethanol, caffeine, lysergic acid dieth yla m id e (L S D ), phencychdine (PCP) and A ^-te tra h yd ro ca n n a b in o l (T H C ; see Schechter & C alcagnetti 1993; T zschentke 1998).

CP P is a re la tive ly sim ple procedure to carry o ut, and has a n u m b e r o f m e th o d o lo g ic a l advantages th a t have made it an extrem ely p o p u la r to o l fo r the analysis o f rew ard. Its m a jo r advantage is th a t i t tests the anim al un d e r study in a d m g -fre e state, thereby a vo id in g the p o te n tia l effects o f the d m g (o r o th e r stim ulus) u n d e r test o n p e rc e p tio n o r m o tiv a tio n . I t can be used in a w id e range o f species, and does n o t require surgery b e fo re starting the e xp e rim e n t, u n lik e se lf-a d rn in istra tio n paradigm s. I t is sensitive to lo w doses o f drugs, and a preference can o fte n be p ro d u ce d fo llo w in g a single p a irin g o f the stim ulus and context. H o w e v e r, the task does have a n u m b e r o f conceptu al drawbacks. T h e m a jo r c ritic is m o f C P P as a measure o f rew ard is based o n the fact th a t the a n im al u n d e r study is required to

Chapter two Materials and methods

fo rm an association betw een the subjective effects o f the stim ulus and the c o n te x t in w h ic h it is adm inistered: the reward-associated c o n te x t then takes o n a secondary re in fo rc in g e ffe ct in its o w n rig h t, causing the anim al to m ake m o re o p e ra n t approaches tow ards the context, o r alter its b e h a vio u r w ith in it, such th a t the tim e spent in the c o n te x t is increased. D rugs th a t in te rfe re w ith learning and m e m o ry processes m ay p re v e n t such associations being made, thereby m asking a p o te n tia l re w a rd in g effect. S im ilarly, an e ffe c t o f the drug u n d e r test o n the a b ility o f an anim al to becom e fa m ilia r w ith a c o n te x t m ay render the drug-paired c o m p a rtm e n t m o re ‘n o v e l’ than the alternate c o m p a rtm e n t, to w h ic h it was exposed w ith o u t the drug. In this case, the anim al u n d e r test m ay m o ve tow ards the drug- associated c o m p a rtm e n t d u rin g the test phase because o f this n o v e lty e ffe c t ra th e r than a p rim a ry re w a rd in g e ffe ct o f the drug un d e r smdy. A fin a l caveat is th a t state-dependent learning m ay occur, such th a t associations m ade betw een the re w a rd in g stim ulus and the co n te x t are expressed o n ly w h e n the anim al is u n d e r the in flu e n ce o f the d ru g stim ulus und e r study. Since testing is usually carried o u t in a drug-free state, such an e ffe ct could m ask a s tim u lu s ’s re w a rd in g e ffe c t d u rin g this task. N evertheless, the draw backs o f the CPP p rocedure are few in com parison to its s im p lic ity and robusm ess. D espite the p o te n tia l co n fo u n d s o f the task, CPP does p ro v id e in fo rm a tio n a b o u t the a ffe ctive value o f m any s tim u li, and unique in fo rm a tio n ab o u t the re w a rd in g e ffe c t o f co n te xtu a l cues associated w ith such s tim u li (see B ardo & Bevins 2000).

In the CPP experim ents presented in this thesis, the re w a rd in g prop e rtie s o f m o rp h in e o r cocaine were assessed in a counterbalanced, unbiased design based o n th a t o f M a ld o n a d o

et

al.

(1997). Preference scores were expressed as the w ith in -s u b je c t d iffe re n ce in tim e spent in the drug-associated co m p a rtm e n t a fte r c o n d itio n in g relative to th a t b e fo re co n d itio n in g . T h e scores o f animals treated w ith d ru g in one o f the tw o co m p a rtm e n ts w ere com pared to those achieved b y vehicle (saline) exposure in b o th co m p a rtm e n ts th ro u g h the c o n d itio n in g p e rio d , to c o n tro l fo r the effects o f stress and exposure to han d lin g , in je c tio n

per se

and the n o v e lty o f the apparatus.

2.5.3.1.

Apparatus

T h e CPP apparatus used in the experim ents presented in this thesis consisted o f tw o c o m p a rtm e n ts (146 m m [1] X 138 m m [w] x 141 m m [h]) separated by a central neutral zone (50 m m [1] X 138 m m [w] x 141 m m [h]; Figure 2.3). A l l apparatus was constructed

Chapter two Materials and methods

fro m w h ite Plexiglas. T he tw o com partm ents d iffe re d in w all pattern and flo o r texture: one c o m p a rtm e n t had black h o riz o n ta l stripes painted on the walls and flo o r, w h ils t the other had black spots painted on the walls and flo o r, and a m etal g rid on the flo o r. A rem ovable g u illo tin e d o o r whose in n e r surface was painted in the same pattern as its co m p a rtm e n t could be used to enclose each end co m partm ent. Clear Plexiglas lids were in place to prevent escape.

Position of guillotine doors

Metal grid Central neutral zone

Figure 2.3 CPP apparatus.

View o f apparatus from above show ing sp o tted and striped co m p artm en ts, position o f the m etal grid, and the central neutral zone. Small arrows indicate the positions o f the rem ovable guillotine doors (not show n), w hich are painted in the same p attern as the c o m p artm en t walls.

2.5.3.2.

Procedure

Chapter two Materials and methods

In document Diseño y Construcción de un Prototipo para Generación de Hidrógeno y Oxígeno (página 50-64)