Contrastación de la hipótesis especifica N°

D-aspartate is transported on glutamate uptake carriers, as shown by radiotracing in

mammalian preparations (Erecinska et a l, 1983), and by electrical recording in

salamander Müller cells (Barbour et a l, 1991) and in Xenopus oocytes expressing, for

example, EAACl transporters (Kanai and Hediger, 1992). However D-aspartate has a

low affinity for non-NMDA receptors (Monaghan et a l, 1984). In Purkinje cells from

12 day old rats, the only ionotropic glutamate receptors are non-NMDA receptors

(NMDA receptors are not expressed after postnatal day 10: Konnerth et a l, 1990;

Perkel et a l, 1990; Momiyama et a l, 1996), so D-aspartate has a suitable

pharmacological profile for selectively activating uptake instead o f channels. By contrast (as shown below) L-glutamate mainly produces a current in the Purkinje cell by activating non-NMDA receptors. Fig. 5. IB shows the membrane current o f a whole-cell clamped Purkinje cell during iontophoresis of D-aspartate onto its soma. D-aspartate evokes an inward membrane current.

How does D-aspartate evoke this current? There are three possibilities (Fig. 5.2). First, D-aspartate might open glutamate-gated channels directly, although D-aspartate is a poor

Fig. 5.1 Application of D-aspartate iontophoretically, to cerebellar Purkinje cells. A. Schematic diagram of iontophoresis experiment. Purkinje cells were whole-cell clamped, and D-aspartate was applied onto the soma by changing the iontophoresis holding current of +20nA to an ejecting current of -40nA. B. lontophoresing D-aspartate evoked an inward current. Pipette solution was solution D, Table 2.2. The holding potential was - 96mV.

Purkinje Cell

Iontophoresis

Pipette

Patch Pipette

Iontophoresis

Current

Uptake

Current

HP -96mV

+ 20p A

-40pA

Fig. 5.2 Diagram o f three possible causes of D-aspartate-evoked current in Purkinje cells.

Top arrow: D-aspartate might directly open the non-NMDA receptor channels in

Purkinje cells. Bottom arrow: D-aspartate might inhibit the uptake of glutamate in

surrounding cells, and raise the extracellular glutamate concentration which leads to

opening o f non-NMDA channels in Purkinje cells. Middle arrow: D-aspartate may be

How does D-asp evoke a current?

Purkinje Cell

Opening o f channels

D-ASP

Inhibit glutamate uptake

raise [glu],,

open channels

Uptake 2Na

«

inhibit uptake in surrounding cells (i.e. glial cells or presynaptic terminals), and thus raise the extracellular glutamate concentration, leading to opening o f non-NMDA channels in the Purkinje cells. Thirdly, D-aspartate may be directly taken up into the Purkinje cells by glutamate uptake carriers. To distinguish these possibilities the following experiments were performed.

Fig. 5.3A and B shows the effect on the current evoked by D-aspartate o f superfusing kynurenate (ImM) which blocks non-NMDA and NMD A receptors (Perkins and Stone, 1985, Stone and Burton, 1988), D-APV (50|iM) which blocks NMDA receptors (Davies

and Watkins, 1982, Evans et a l, 1982), and TTX (l|iM ) which blocks voltage-gated

sodium channels (reviewed by Mosher, 1986). This cocktail o f blockers had little effect on the D-aspartate-evoked current; in 19 cells it reduced the current by 19±6 (S.E.M.) %. Even superimposing 50p.M CNQX on top of the cocktail of kynurenate, D-APV and TTX had little effect on the D-aspartate-evoked current (Fig. 5.3C). In 19 cells the current was reduced by a further 11±5%. The relatively weak effect o f antagonists to non-NMDA and NMDA receptors suggests that most of the current evoked by D- aspartate does not result from the opening o f glutamate-gated ion channels. Fig. 5.3D and E show, however, that the glutamate analogue PDC (300p.M), which is known to

block glutamate uptake (Bridges et a l, 1994), reversibly reduced the current when

applied on top o f the channel blockers mentioned above. In 20 cells the current was reduced by 71 ±4%, suggesting that D-aspartate generates a current by binding to the glutamate uptake carrier. Purkinje cells can also show aspartate-evoked currents caused by glutamate, released by aspartate-glutamate exchange on uptake carriers, activating non-NMDA receptors (Renard and Crepel, 1996), or caused by aspartate activating a

Ca^^-permeable channel (Yuzaki et a l, 1996). Both o f those currents were abolished by

CNQX and so do not contribute significangly to the D-aspatate-evoked current I observed (Fig. 5.3C).

By contrast, when L-glutamate was iontophoresed instead o f D-aspartate, the blockers kynurenate, APY and TTX reduced the current by 76±6 (S.E.M.) % (6 cells), and 50p.M CNQX reduced the current remaining in these blockers by 93±3% (2 cells). These

Fig. 5.3 Pharmacology of the current evoked by iontophoresis o f D-aspartate (bar) onto whole-cell clamped Purkinje cells in cerebellar slices. A-C. Effect of glutamate receptor

blockers. A. Control response with lOOpM picrotoxin present to block GABAa

receptors. B. Superfusing ImM kynurenate, SOpM D-APV and IpM TTX on top of the

picrotoxin in A. C. Superfusing 50pM CNQX on top of the blockers in B. D, E. Effect

of the glutamate uptake carrier blocker PDC. D. Superfusing 300|iM PDC on top of the

blockers in C. E. recovery from D. A1 traces were taken from the same cell. Pipette

A

_PTX

B

_+KYN+APV+TTX

20pA

4s

results suggest that the glutamate-evoked current is generated largely by the activation of non-NMDA channels, and confirm that the doses of blockers used would have greatly reduced the response to D-aspartate if it had been produced by glutamate-gated channels.

The need for a further set of control experiments was suggested by the work of Linden et

a l (1994), who observed inward currents in cerebellar Purkinje cells when glutamate

analogues activating metabotropic glutamate receptors were applied. They suggested that the metabotropic agonists raised [Ca]i which activated the NaVCa^^ exchanger and thus generated an inward current (since 3 Na^ enter the cell on this exchanger for each

Ca^^ that leaves the cell). To investigate the possibility that D-aspartate activates

metabotropic receptors and thus generates a NaVCa^^ exchange current, the following experiments were carried out (in the presence o f lOOpM picrotoxin, ImM kynurenate,

50pM D-APV and 50|iM CNQX). Application o f 500pM (+)-a-methyl-4-

carboxyphenylglycine (MCPG), an antagonist to the mGluRi type o f metabotropic

receptor (Ito et a l, 1992, Jane et a l, 1993, Collingridge and Watkins, 1994) found in

Purkinje cells (Batchelor et a l, 1994), had no effect on the D-aspartate evoked current

(reduced by 7±11% in 3 cells: Fig. 5.4A). Similarly application of lOOpM (1S,3R)-1-

aminocyclopentane-1,3 -dicarboxylic acid {trans-ACPT>), a metabotropic receptor

agonist, did not alter the current (increased by 4±3% in 4 cells: Fig. 5.4B), although one

might have expected this dose of trans-ACVT) to nearly saturate the metabotropic

receptors (East and Garthwaite, 1992; Palmer et a l, 1989) and thus reduce any current

produced by D-aspartate acting on those receptors. To test further the possible

involvement o f the NaVCa^^ exchanger, lOOjuM dichlorobenzamil and 200pM La^^,

putative exchanger blockers (Andreeva et a l, 1991; Plasman et a l, 1991; Niggli and

Lederer, 1993), were applied. Neither of them had any effect on the current (reduced by 5±12% in 4 cells in the presence of dichlorobenzamil, and increased by 5±8% in 5 cells in the presence of La^^: Fig. 5.5).

The D-aspartate evoked current was also unaffected by Ba^^ (Fig. 5.6), ruling out the possibility that K^, which could be released from glial cells depolarised by D-aspartate uptake, generates the current by passing through inward rectifier channels (which have a

Fig. 5.4 Effect of a metabotropic receptor blocker and agonist on the current evoked by

D-aspartate (bar). A. {Left) Response to iontophoretically applied D-aspartate. {Right)

Response to D-aspartate in the presence of the metabotropic glutamate receptor blocker

MCPG (500pM). Pipette solution contained CIO4 (solution F, Table 2.2) to increase the

carrier-generated current as described in section 5.6. B. {Left) Response to

iontophoretically applied D-aspartate. (Right) Response to D-aspartate in the presence of

the metabotropic glutamate receptor agonist trans-AC¥D (lOOpiM). Pipette solution

A

MCPG

200pA

4s

B

50pAj__

4s

Fig. 5.5 Effect of Na/Ca exchange blockers on the current evoked by D-aspartate (bar).

A. {Left) Response to iontophoretically applied D-aspartate. (Right) Response to D-

aspartate in the presence o f 100 pM dichlorobenzamil (DCB). Pipette solution contained

CIO4’ (solution F, Table 2.2) to increase the carrier-generated current (see section 5.6).

B. (Left) Response to iontophoretically applied D-aspartate. (Right) Response to D-

aspartate in the presence of 200pM lanthanum (La^^). Pipette solution was solution D, Table 2.2. The holding potential was -96mV.

A

DCB

lOOpA

4s

B

5 Op A

4s

Fig. 5.6 Effect o f the inward-rectifying potassium channel blocker (Ba^^) on the current

evoked by D-aspartate (bar). {Left) Response to iontophoretically applied D-aspartate.

(Right) Response to D-aspartate in the presence of 6mM barium (Ba^^) (solution C,

Table 2.1). Pipette solution was solution D, Table 2.2. The holding potential was

50pA

voltage-dependence similar to that of the current evoked by D-aspartate, see below; Newman, 1985).

These pharmacological data suggest that the current is generated by D-aspartate being taken up into Purkinje cells by glutamate transporters (with the inward current being generated partly by co-transported Na^ ions and partly by Cl' movement through an anion channel in the carrier’s structure, as described below).