RELACIONES DE LARGO PLAZO DEL PRODUCTO BRUTO INTERNO

F ig u re 5.23 T ra n s ie n ts in th e soma of a P u rk in je ceil m onitored w ith 1.2mM Fluo-3

The fig u re show s lig h t tr a n s ie n ts from th e soma of a P u rk in je cell v o ltag e clamped w ith a p a tc h p ip e tte co n tain in g 1.2mM Fluo-3 an d 50pM caged InsPg. P h otolytic re le a se of 9pM InsP^ was cap ab le of mobilising calcium d e sp ite th e in c re a se d b u ffe rin g (2400:1; bound to f re e calcium a t O.lpM fre e calcium) in tro d u c e d in th e cell by Fluo-3.

tis s u e . F ir s t it may be t h a t th e en d o g en o u s b u ffe rin g of P u rk in je cells tig h tly re g u la te s th e f r e e calcium c o n c e n tra tio n so th a t much h ig h e r c o n c e n tra tio n s of InsPg a r e re q u ire d to in c re a se th e fre e calcium c o n c e n tra tio n to lev els n e c e s sa ry fo r c o o p erativ e action of calcium on th e InsPg re c e p to rs . D espite th e high calcium b u ffe rin g cap acity th o u g h t to e x ist in th e P u rk in je cells (Vill e t al, 1991), th is seems to be an unlikely ex p lan atio n on th e b asis of th e la rg e ra n g e of calcium b u ffe rin g u sed in th e ex p erim en ts d e sc rib e d h e re (F ig u re 5.23); th e calcium b u ffe rin g in tro d u c e d by SOOpM F u r a p tr a (11 bound to 1 fre e a t O.lpM fre e calcium) is small, an d t h a t in tro d u c e d by 1.2mM Fluo-3 (2400 bound to 1 fre e a t O.lpM f re e calcium) is la rg e com pared to th e end o g en o u s b u ffe rs (eg 75 bound to 1 f re e in chrom affin cells, N eher an d A u g u stin e 1992). The second p o ssib ility is th e p re se n c e of hig h d e n s ity of in activ e InsPg b in d in g site s w hich r e s u lt in a ra p id re d u c tio n of [InsPg] a d ja c e n t to th e calcium re le a se s ite s . A lte rn a tiv e ly th e th ird p o ssib ility is th a t th e fu n ctio n a l InsPg r e c e p to rs in th e P u rk in je cells a r e d iffe re n t from th o se p re s e n t in th e p e rip h e ra l tis s u e . In d eed in s u p p o rt of th is h y p o th e sis a lte rn a tiv e sp licing an d tis s u e sp e c ific e x p re ssio n of th e m essen g er RNA fo r th e InsP^ re c e p to r h a v e b een r e p o rte d (M ignery e t al, 1990; Nakagawa, 1991; Danoff e t al,

1991; Ross e t al, 1992). F u rth e rm o re th e m essenger RNAs a lte rn a tiv e ly sp liced v a r y in th e amino acid se q u en c es encoding for th e SI o r SII re g io n s, which a r e th o u g h t to be im p o rtan t in th e binding of InsPg, and p h o sp h o ry la tio n of th e InsPg re c e p to r (Nakagawa e t al, 1991; Danoff e t al,

1991), an d may d eterm in e th e p o ten cy of InsP^ in calcium re le a se (Nakagawa e t al, 1991).

W hatever th e re a so n fo r th e h e te ro g e n e ity of InsP^ action in th e P u rk in je cells w ith th a t of o th e r tis s u e s s tu d ie d , it is a p p a re n t th a t th e re q u ire m e n t fo r f a s t sig n allin g in n e u ro n e s h as re su lte d in a system in w hich a n in c re a se in th e cytosolic c o n c e n tra tio n of InsPg by stim ulation of G p ro te in linked n e u ro tra n s m itte r r e c e p to rs is cap ab le of ra p id ly in c re a sin g th e [Ca^*].. The elev atio n of [Ca^*]. in th e s h o rt term modifies th e e x c ita b ility of th e membrane by a c tiv a tin g th e calcium g ated potassium c h a n n e ls, an d in th e long term s e ts in action calcium a c tiv ate d p ro c e sse s s u c h a s g en e tr a n s c r ip tio n (eg Morgan an d C u rran , 1988), an d p ro te in s y n th e s is (eg B rostorm an d Brostorm , 1990).

In e x p erim en ts d esc rib e d h e re th e ch a n g e s in th e flu o re sc e n c e em itted by th e Ccdcium in d ica to r from a defin ed reg io n of th e cell was m easured w ith a p h o to tu b e allowing a time, b u t n ot sp a tia lly reso lv ed s tu d y of th e c h a n g e s in th e [Ca^*]^. The p re se n c e of calcium g ra d ie n ts a t d iff e r e n t p a r ts of th e cell could n o t be d e te c te d . S uch g ra d ie n ts may in d eed e x ist an d could play an im p o rtan t physiological role. A lthough

im aging te c h n iq u e s p ro v id e sp a tia l reso lu tio n , to d a te th e ir com bination w ith flu o re s c e n t in d ic a to rs h ave n o t allowed a time reso lv ed (ms re so lu tio n ) s tu d y of [Ca^*]j . In th e view of th e fa c t th a t th e c h a n g e s in th e [Ca^*]. r e p o rte d h e re a r e extrem ely f a s t th e p r e s e n t imaging te c h n iq u e s would be u n a b le to p ro v id e an a c c u ra te indication of th e ch an g e s in th e [Ca^*]. .

A lthough th e elev atio n of [Ca^^Jj in th e P u rk in je cells by InsPg is a compliment of th e p ro p o sed action of glutam ate m etabotropic r e c e p to rs th o u g h t to be p r e s e n t in th e s e cells (eg Llano e t a l 1991), f u r t h e r ex p erim en ts a re re q u ire d to d em o n strate its role in s y n a p tic tra n sm issio n . For th is p u rp o se th e th in slice p ro v id e s a good p re p a ra tio n b e ca u se a p ro p o rtio n of th e s y n a p tic in p u t to a cell in a slice c u t a t th e a p p ro p ria te o rie n ta tio n is le ft in ta c t. T hese p athw ays may be stim ulated in th e p re s e n c e of io n otropic re c e p to r b lo ck ers and th e ch a n g e s in [Ca^*]. m onitored. F u rth e rm o re th e e ffe c t of InsP^ released photo ly tically on actio n p o te n tia ls evoked sy n a p tic a lly can be stu d ie d . P a rtic u la r a tte n tio n sh o u ld be g iv en to th e c h a n g es in th e [Ca^*]. of th e d e n d rite s becau se th e h ig h m em brane s u rfa c e a re a to volume may r e s u lt in a lte re d k in e tic s of calcium re le a se and removal. However th e s tu d y of InsP^ action in th e d e n d rite s is d iffic u lt due to th e poor sig n al to noise ra tio of th e flu o re sc e n c e em itted by th e calcium in d ic a to rs to th e b ack g ro u n d flu o resc e n ce in th is reg io n . Longer ’cell lo ad in g ’ time, and p e rh a p s b rig h te r calcium in d ic a to rs a r e r e q u ire d so th a t th e ch a n g es in [Ca^^]. may be stu d ie d in th e d e n d r ite s w ithout d is tu rb in g in tra c e llu la r calcium b u ffe rin g .

A lthough th e p re se n c e of exceptionally high c o n c e n tra tio n s of InsPg b in d in g s ite s and calcium b in ding p ro te in s made th e P u rk in je cells th e obv io u s c a n d id ate fo r th e s tu d y of InsP^ action in n e u ro n e s, P u rk in je cells may not be a good re p re s e n ta tiv e of th e InsP^ fu n ctio n in th e CNS fo r th e same re a so n s. F u rth e rm o re s u b s ta n tia l d iffe re n c e s in th e levels of th e a lte rn a tiv e ly sp liced form s of th e InsP^ re c e p to r o ccu r in th e b rain ; one form w ith 15 amino acid s d eleted acco u n ts fo r 75-85% of InsP^ r e c e p to rs in th e cerebellum b u t only fo r 12% of re c e p to rs in th e c e re b ra l c o rte x (Nakagawa e t a l 1991). T h e re fo re f u r th e r s tu d ie s a r e re q u ire d to c h a ra c te riz e p ro p e rtie s of InsP^ sig n al tra n sd u c tio n in n e u ro n e s o th e r th a n P u rk in je cells to e s ta b lis h w h eth er th e ad ap tatio n se e n in P u rk in je cells is a g e n e ra l f e a tu re of all n e u ro n es.

InsPg re le a se d photo ly tically in th e cytosol of c u ltu re d n e u ro n e s did not mobilise calcium. One explanation may be th a t th e n e u ro n e s s tu d ie d , hippocam pal pyram idal cells, s tr ia ta l n eu ro n es, c e re b e lla r g ra n u le cells, an d DRGs do n ot h av e fu n ctio n a l InsP^ re c e p to rs , o r th e r e c e p to rs a r e d iffe re n t from all o th e r s s tu d ie d . This, a lth o u g h p o ssib le on th e b a sis of th e e x iste n c e of a lte rn a tiv e ly sp liced form s of InsP^ re c e p to r mRNAs, is th e

le ss lik ely ex p lan atio n if th e p o ssib ility of ’c u ltu re a r tif a c t’ is co n sid e red . Num erous a lte ra tio n s in th e physiological p ro c e sse s of cells k e p t in c u ltu re h av e b een re p o rte d . For example in c u ltu re d P u rk in je n e u ro n e s no a lte ra tio n in th e [Ca^*]. could be d e te c te d when th e m etabotropic r e c e p to rs w ere stim u lated (B rorson e t al, 1991). These cells how ever e x p re sse d h ig h I n s?3 re c e p to r b in d in g s ite s an d m anifested a n in c re a se in [Ca^^]. w hen th e y w ere d e p o larised , o r stim ulated with caffein e o r glutam ate in th e a b se n c e of g lu tam ate ch an n el b lo ck ers (B rorson e t al, 1991). In c o n tr a s t to th e c u ltu re d P u rk in je n e u ro n e s an in c re a se in in tra d e n d ritic [Ca^^]. of P u rk in je cells in th in slices of th e cerebellum following stim ulation of th e g lu tam ate m etabotropic r e c e p to rs h a s been r e p o rte d (Llano e t al, 1991). In hippocam pal pyram idal cells a ’m etabotropic’ calcium m obilising action of g lu tam ate could only be d e te c te d if a d ep o larisin g ’c o n d itio n in g ’ p u lse was u sed to ’fill u p ’ th e s to re s (M urphy an d Miller, 1988). The n a tu re of th e s e a r tif a c ts a r e n o t clear, b u t could a ris e from th e lack of p ro p e r d iffe re n tia tio n of th e s e cell in c u ltu re in th e ab se n c e of th e norm al s y n a p tic in p u ts. In o rd e r to avoid th e possible ’c u ltu re a r tif a c ts ’, f u r t h e r ex p erim en ts on n e u ro n es in slices obtain ed from v a rio u s reg io n s of th e b ra in sh o u ld be done to ex p lo re th e InsPg fu n c tio n in th e se cells.

C hapter 6 O verview

6. O verview

E xperim ents w ere d esc rib e d d u rin g which p o s ts y n a p tic a c tio n s of