A n a lte rn a tive strategy w o u ld be to engineer lines o f m ice th a t lack N K l receptor expression in specific regions o f th e ir brain, and to com pare th e ir b e h a vio u r in m o rp h in e re w a rd -re la te d paradigm s to th a t o f N K l'^ ' m ice. T h is co u ld th e o re tica lly be achieved using the bacteriophage P I-d e riv e d
C re/loxP
re c o m b in a tio n system (Tsienet ai
1996a). T h is process in vo lve s crossing transgenic m ice th a t express Cre recom binase u n d e r the c o n tro l o f a p ro m o te r active w ith in a specific b ra in re g io n w ith a second transgenic m ouse Hne in w h ic h one o r m o re o f the exons o f the gene o f in te re st has been fla n ke d by tw oloxP
sites. I n the o ffs p rin g o f such a cross, the C re recom binase catalyses re c o m b in a tio n betw een th e tw o fla n k in gloxP
sites, thereby excising the gene o f interest, solely in those cells th a t express Cre in the parental m ouse line. T h is procedure has already been used in m ouse b e h a vio u ra l studies to exam ine the effects o f d e le tio n o f the N -m e th yl-D -a sp a rta te ( N M D A ) re c e p to r s u b u n it 1 fro m the C A l (Tsienet al
1996b) o r C A 3 (Nakazaw aet al
Chapter six Behavioural ejfects o f ablation o f neurones expressing the neurokinin-1 receptor
2002) regions o f the hippoca m p us o n learning and m e m o ry behaviours. H o w e ve r, the p ro d u c tio n o f such lines o f m ice is extrem ely tim e -co n su m in g and expensive, and therefore im p ra c tic a l fo r the co m p a riso n o f num erous b ra in regions.
6.2.1.3. Ablation of neurokinin-1 receptor-expressing neurones
T h e approach taken in these experim ents was to use the n e u ro to x in SP-SAP (W iley & L a p p i 1997). T h is is a conjugate o f SP w ith the rib o s o m e -in a c tiv a tin g p ro te in saporin (SAP) fro m the seeds o f the p la n t
Saponaria officinalis
(L a p p iet a l
1985; S tirpeet al
1983, 1992). Since SAP c a n n o t cross the plasma m em brane, the o n ly w ay by w h ic h it can get in to a cell and lead to its death is b y ‘p iggyba cking’ o n to SP. SP, w h e n it binds to the N K l receptor, is ra p id ly internalised along w ith the receptor, and enters the endosom al c o m p a rtm e n t. T h e fa ll in p H w ith in the endosom e causes th e disso cia tio n o f the ligand fro m its receptor: SP is subsequendy degraded in the lysosom e w h ils t its re ce p to r is recycled back to the m em brane, w here it can b in d ligand once m o re (F igure 6.1 A ; G a rla n det al
1994; G ra d yet al
1995; M a n ty het ai
1995a,b; S o u th w e llet a i
1996). C o n ju g a tio n o f SAP to SP allow s the to x in to enter the cell, reach the ribosom es, and causing th e k in a c tiv a tio n , and eventual cell death (Figure 6 .IB ). Since SP-SAP is o n ly intem ahsed by those cells expressing N K l receptors, the to x in is specific fo r this cell p o p u la tio n , leaving cells th a t do n o t express the re ce p to r in ta c t (M a n tyhet ai
1997; W ile y & L a p p i 1997).Chapter six Behavioural effects o f ablation o f neurones expressing the neurokinin-1 receptor
SP
NK1R
Endosome
Lysosome
SP-SAP
NK1R
Endosome
Lysosome
@ Ribosome
inactivation
& cell death
Figure 6.1 Internalisation o f SP and SP-SAP. A:
Upon binding to the N K l receptor (1), SP is
internalised via clathrin-coated pits (2) to the endosomal compartment (3), where the decrease in
pH causes the receptor and ligand to dissociate. SP is degraded in the lysosome (4), while the N K l
receptor is recycled back to the synaptic membrane (5).
B:Conjugation of SAP to SP allows the
SAP toxin to ‘piggyback’ into the cell. SP-SAP follows the same route as SP into the cell (1-3) and
travels, possibly via the lysosome (4), to inactivate ribosomes and eventually cause cell death (5).
Ihe N K l receptor is recycled to the synaptic membrane as normal (6). NKIR; N K l receptor; SP:
substance P; SP-SAP: substance P-saporin.
T he use o f SP-SAP therefore allow s the selective a b lation o f cells expressing the N K l receptor w ith in a specific area o f tissue. T h is represents a d iffe re n t approach to the effects o f genetic m a n ip u la tio n o r pharm acological blockade o f the receptor, since this m e th o d brings about d e stru ctio n o f entire cells - in the case o f neurones, N K l receptor-expressing
Chapter six Behavioural ejfects o f ablation o f neurones expressing the neurokinin-1 receptor
cells also express o th e r receptors and neurotran sm itte rs, and m ay have num erous connectio ns w ith o th e r neurones. A b la tio n o f N K l receptor-expre ssing neurones th e re fo re brings a b o u t the a d d itio n a l re m o va l o f these co n n e ctio n s and the re ce p to r and n e u ro tra n s m itte r systems coexpressed w ith the N K l receptor. A lth o u g h the ne u ro ch e m ica l effects o f N K l re ce p to r gene k n o c k o u t o n these cells has n o t been th o ro u g h ly investigated, i t is clear th a t N K l ’^' m ice s till possess the cells w h ic h express the re ce p to r in w ild type m ice (D e F elipe
el al.
1998). T h e use o f SP-SAP th e re fo re m ay b rin g ab o u t m o re dram atic changes in lo ca l c irc u itry than genetic loss o f the re ce p to r, albeit life lo n g , and certa in ly than its acute o r c h ro n ic antagonism . N evertheless, this approach does fo rm a co m p ro m ise betw een e xperim enta l s im p lic ity and an accurate assessment o f the ro le o f the substance P / N K l re ce p to r system in the c o n tro l o f b ehavio ur.6.2.1.3.1. Previous studies using substance P-saporin
SP-SAP has aheady been used to dem onstrate a ro le fo r N K l receptor-expressing neurones in la m ina I o f the spinal c o rd in the transm ission o f hyperalgesia (M a n tyh
et al.
1997) and c h ro n ic p a in signals (N ic h o lset a l
1999), as w e ll as a ro le fo r superficial m edullary and cervical dorsal h o rn cells in the ora l irrita tio n m ediated b y capsaicin (Simonset al
2002). A d d itio n a lly , d e stru ctio n o f N K l receptor-expressing cells in lam inae V I I and X o f lu m b a r segments 3 and 4 o f the spinal co rd using a sim ila r c o m p o u n d b ro u g h t about deficits in ejaculatio n [P ru itt & C o o le n 2002), w h ils t a b la tio n o f N K l receptor-expressing neurones in the ro s tra l v e n tro la te ra l m edulla suggested th a t they play a ro le in the c o n tro l o f re sp ira to ry rh y th m and b lo o d pressure (W anget a l
2002). A lth o u g h there have been no pub lish e d studies exarnining the b ehavio ural effects o f a b la tio n o f these cells in the mouse, the experim ents described here were carried o u t in o rd e r to investigate the ro le o f these cells in specific b ra in regions in m o rp h in e rew ard-related behaviours.6.2.2. Experimental approach
T w o b ra in regions w ere chosen fo r analysis in th is study. A lth o u g h there are num erous p o te n tia l sites o f a ctio n o f the N K l re ce p to r in m e d ia tin g m o rp h in e re w a rd behaviours, the nucleus accum bens (N A c c ) and amygdala were selected fo r a p re lim in a ry investigatio n. B o th o f these regions express N K l receptors (see C h apter 3), and have been im p lic a te d in the m e d ia tio n o f rew a rd behaviours (see C hapter 1). H o w e v e r, in this study n o a tte m p t
Chapter six Behavioural ejfects o f ablation o f neurones expressing the neurokinin-1 receptor
was m ade to b rin g ab o u t a b la tio n selectively in the core o r shell regions o f the N A c c o r in the various subnucle i o f the amygdala. H o w e v e r, this m o re general a pproach was used as a firs t a tte m p t at re p lic a tin g the behaviours o f N K l '^' m ice fo llo w in g N K l rece p to r- expressing cell ablation , w ith the aim o f iso la tin g the site o f a c tio n o f the SP / N K l re ce p to r system to one o f these tw o regions o f the m ouse brain. T h e effects o f cell abla tio n o n anxiety behaviours w ere also assessed in these m ice.
6.3. General materials and methods
M u c h o f the w o r k presented in this chapter was carried o u t as a jo in t study w ith P atricia M u rtra , a P h D student based at the U niversitas M ig u e l H ernande z, A lic a n te , Spain. T he w o rk described in the fo llo w in g sections was d iv id e d equally betw een D r . M u rtra and m yself: 6.4.1; 6.4.2; 6.4.3; 6.4.5; and 6.5.1.2.
6.3.1. Stereotaxic surgery
Stereotaxic surgery was used to de live r com pound s to specific regions w ith in the brains o f m ice. T h is techniqu e in vo lve s the stabilisation o f the anaesthetised m ouse’s head w ith in a fram e. F o llo w in g exposure o f the skull, the p o s itio n o f la n d m a rk jo in ts in the skull (Bregm a and / o r L am bda; see F igure 6.2) are measured, fr o m w h ic h the p o s itio n o f the targets w ith in the b ra in can be calculated o n the basis o f a b ra in adas (F ra n k lin & Paxinos 1997). A syringe co n ta in in g the substance to be in je cte d is attached to a m anoeuvrable h o ld e r, fix e d to the stereotaxic fram e, w h ic h can be m o ve d separately in three dim ensions b y way o f calibrated screws. T h is fine m o ve m e n t o f the syringe allow s the tip o f the needle to be p o s itio n e d at a precise sp o t (0.1 m m accuracy) w ith in the b ra in re la tive to the skull jo in t.
Chapter six Behavioural ejfects o f ablation o f neurones expressing the neurokinin-1 receptor
Sagittal suture
Lambdoid suture
Bregma
Lambda
F ig u re 6.2 M ouse sku ll. V iew o f m ouse skull from above show ing sagittal and lam bdoid sutures, and the positions o f Bregm a and Lam bda. L am bda is the p o in t o f intersection o f the projection lines o f b est fit through the sagittal and lam bdoid sutures.