ID REQUERIMIENTO DESCRIPCIÓN PRIORIDAD DIFICULTAD

Cha^tei^^^Metju)ds^an (i) 5 00 r 100 4 0 0 - 80 3 00 - 60 - 40 100 - 20 0 20 40 60 60 100 120 600 -, 100 500 - 60 60 300 - 4 0 - 20 1 0 0 - 4 23 W a s h T i m e ( s e c o n d s ) TE MP ER A TU RE C O (iii) 1000 r 100 6 0 0 - 60 60 o 2 0 0 - : 20 0 20 40 60 6 0 0 -, r 100 5 0 0 - - 60 w - 60 300 - i 3 O CD 2 0 0 - 4 0 > - 20 100 - 4 23

Incubation Time (minutes) T E M P ER A TU RE C O

(v) 1 0 0 1 _80 - < 1- O I- 60 - O a . (/) 0 -I

C h a ^ te r^ ^ ^ eth o d ^ a n ^ M ^ e n n h

2.3.9 A lte r a tio n s in GABAg a n d G lu ta m a te B in d in g S ite s R e s u ltin g

from N e o n a ta l C a p sa icin T r ea tm en t

(a) GABAg Sites

(i) B inding Procedure

As described above, 50mM Tris-HCl + 2.5mM CaClg (pH=7.4 a t room tem p eratu re) was used. B inding w as carried out a t room tem perature (-20 ± 2°C) for 20 m inutes w ith 40pM isoguvacine (to block GABAa sites) and [^H]-GABA (50-200nM ; spec. act. = 87.3Ci/mmol). Non-specific binding w as done in th e presence of lOOpM (-)-baclofen (to block th e GABAg sites). The w ash procedure followed was as described in Section 2.3.7(a).

(ii) Q u an titativ e A utoradiography

Following fourteen days of exposure, th e photographic im ages on th e em ulsion coated coverslips were developed an d the tissue sections stain ed as described in Section 2.3.3(c).

(b) G lu tam ate Sites

(i) O ptim isation Studies

The binding param eters cited in the literatu re for autoradiographic studies of the ionotrophic g lu tam ate sites are extrem ely varied.

Compared to the brain, th ere have been relatively few rep o rts on intact spinal cord sections. Therefore, binding studies were carried out on the spinad cords from naïve ra ts to determ ine th e optim um binding conditions.

Cha^tei^2^^^J4ethod^ndMa^

Initial binding conditions (see Table 2(b)) used were based on those previously reported (M onaghan et al., 1984; Bowery et al., 1988; S h a w al., 1991,1994; Henley al., 1993; Shaw a n d Ince, 1994). E ssen tially , the procedures for determ ining th e best binding conditions for these ligands are as described in Section 2.3.8(a). The results obtained from th e various investigations are show n in F ig u res 2(j), 2(k) an d 2(1).

(ii) B inding Procedure

Binding studies were carried out as described in Section 2.3.2. The binding param eters and incubation solutions used for th e stu d y of th e th ree g lu tam ate binding sites were as outlined in Table 2(c).

AMPA receptor binding sites were studied w ith Tris-H Cl (50mM + 2.5mM CaClg) containing [^H]-AMPA (10-200nM ; spec. act. = 53.0Ci/m m ol) an d lOOmM KSCN. The chaotropic ion, SON’, appears to shift the receptor to a high affinity sta te (N ielsen et al.,

1988; H onoré et al., 1989; W atkins et al., 1990) so facilitatin g the b in d in g process. Non-specific binding w as carried out in the presence of lOOpM quisqualic acid.

The incubation solution used for the dizocilpine binding sites was T ris-H C l (50mM + 2.5mM CaClg) w ith lOmM glutam ic acid an d lOmM glycine added. The pre-incubation w ashing solution did not, however, have any glutamic acid or glycine added. [^H]-dizocilpine (10-40nM ; spec. act. = 22.5Ci/mmol) w as added to all the in cu b atin g solutions while lOOpM of th e 'cold’ ligand w as used to q u an tify non-specific binding.

Cha^ter^2j^^ethodsm^

T ris-acetate (50mM) was used in stead of Tris-H C l + CaClg w hen th e KA receptor binding sites were studied. C alcium shifts th e receptor to its low afihnity sta te (Honoré et al., 1989 an d W atkins

et al., 1990) and hence inhibits binding a t th e hig h affinity sites (B eaum ont et al., 1979; B raitm an an d Coyle, 1987; K rosgaard- L arse n et al., 1991). Studies were carried out in th e presence of

10-200nM [^H]-kainic a d d (spec. act. = 58.0Ci/mmol). Non-specific binding was determ ined w ith lOOpM kainic acid.

(iii) Q u an titativ e A utoradiography

D ried sections were apposed to em ulsion-coated coverslips as described in Section 2.3.3(b). The photographic im ages on the emulsion coated coverslips were developed an d fixed a fte r 12 to 13 days (AMPA and KA) or 15 to 16 days (dizocilpine) of exposure.

Chaptet' 2 : Methods and M aterials

F ig u r e 2(j) O p tim u m b in d in g c o n d itio n s fo r th e b in d in g o f [^H]-AMPA to A M PA s it e s in r a t lu m b a r s p in a l c o r d s e c tio n s .

Results were obtained by the method of liquid scintillation spectrometry (see Section 2.3.8(a) for details). The specific activity of PH]-AMPA was 53.0Ci/mmol[ Total (▲....▲) and non-specific binding (T —T) are expressed as disintegrations per minute (d.p.m.) for three sections. Experiments were carried out in triplicate (n = 3). Specific binding (# —# ) was calculated as the difference between these two values and is expressed as a percentage of total binding.

(i) Wash Time

Specific binding peaked at 10s and appeared to level off at longer wash times.

(ii) Wash Temperature

The specific binding obtained at the two temperatures were similar. (iii) Incubation Time

Specific binding for incubation times of 40min yielded the highest degree of specific binding.

(iv) Incubation Temperature

The specific binding obtained at the two temperatures were similar. (v) Buffer pH