Increase in ASP concentration (expressed as area under the curve,pmol) in striatal dialysate after topical application of drugs. Data were analysed using ANOVA followed by either the Mann Whimey U test or the LSD test as appropriate. * = P < 0.05 compared to vehicle effect; + = P < 0.05, compared to the NMDA effect.
(PBS = phosphate buffered saline; NM 2mM = NMDA 2mM; NM 20mM = NMDA 20mM; NM20TTX = NMDA 20 mM + tetrodotoxin 10 /xM; NM20Cafr = NMDA 20 mM, dialysis with Ca^'*' -free dialysis fluid; High K = 100 mM potassium).
Fig 3,10
PBS
NM 2mM NM 20mM NM20TTX NM20Cafr High K
Increase in Glu concentration (expressed as area under the curve,pmol) in striatal dialysate after topical application of drugs. Data were analysed using ANOVA followed by either the Mann
Whimey U test or the LSD test as appropriate. * = P < 0.05 compared to vehicle effect; + = P < 0.05, compared to the NMDA effect.
(PBS = phosphate buffered saline; NM 2mM = NMDA 2mM; NM 20mM = NMDA 20mM; NM20TTX = NMDA 20 mM + tetrodotoxin 10 /xM; NM20Cafr = NMDA 20 mM, dialysis with Ca-^ -free dialysis fluid; High K = 100 mM potassium).
3.5 DISCUSSION
Basal concentrations of the excitatory amino acids glutamate and aspartate, detected with striatal microdialysis and HPLC were similar to those found by Obrenovitch & Richards, (1994), who used similar experimental conditions (same dialysis probe and anaesthesia, same brain area) as those described in this thesis.
The main finding is that topical application of NMDA increased striatal concentrations of both aspartate and glutamate. In a previous study Palmer et al., (1989) observed that glutamate release
was only significantly increased when a cocktail of GABA-antagonists was added to NMDA. It seems likely that differences between that study in this one are the result of the methodological changes made.
Differences with the previous study are: 1) A different microdialysis probe was used. 2) The flow rate was reduced from 2 to 1 //l/min.
3) Stabilisation time after implantation of the probe was increased from 0.5 to 1 hour.
4) Different anaesthesia was used (chloral hydrate in the previous study, halothane in a mixture of oxygen and nitrous oxide in this study).
5) A feedback temperature controller was used in the present study, ensuring a constant core temperature during the complete experiment. In the previous study a heating mat was used, with no feedback control.
In view of these factors it is not unexpected that in the present study less variability of the
response occurred and that more subtle differences between drug effects can be detected. Also in the present study NMDA released relatively more aspartate than glutamate (20 mM NMDA increased aspartate release by 500 % while it increased glutamate release by 380 %). The reason
this is the case is suggested by a study showing that 30 nmoles of NMDA in 3 fx\ causes extensive damage to the cortex (Palmer et al., 1993). This excessive stimulation may lower extracellular concentrations of glucose, which may be accompanied by a relative increased secretion in ASP over GLU (Szerb & O ’Regan, 1987)
It could be argued that application of NMDA to the surface of the cortex would cause a spreading depression of Leao, characterised by loss of cellular ionic homeostasis and a drastic redistribution of ions between the extracellular and intracellular compartments (Leao, 1944), which would then spread through to the deeper layers. Thus, increased release of amino acids could be the result of a nonspecific effect, rather than being the result of an interaction between drugs applied and receptors on the cell bodies of layer V pyramidal neurones. The main reason that this is unlikely is the delay of approximately 30 to 40 min observed between application of
drugs and the maximal release of neurotransmitters in the striatum. In vitro experiments show that the delay can not be attributed to the dead volume of the microdialysis system. In addition, spreading depression is not mediated by voltage-gated sodium channels (Tobiasz & Nicholson, 1982; Aitken et al., 1991), but in the present study, tetrodotoxin significantly reduced the effect of topically applied drugs. Furthermore a previous study using this paradigm showed that infusion of drugs into frontal cortex had a similar effect as topical application of the drugs to the same frontal region (Palmer et al., 1989). Application of 100 mM potassium to the surface of the cortex, did not affect striatal concentrations of either aspartate or glutamate. The rationale behind
the experiments with 100 mM was the difference in diffusibility into brain tissue between NMDA and potassium. While potassium will be quickly taken up both by neurones and glial cells, NMDA will be taken up only at a low rate by low-affmity transport (Skerrit & Johnston, 1981;
Garthwaite, 1985). Thus it can be postulated that although both potassium and NMDA will depolarise the surface of the cortex when topically applied, only NMDA will be able to diffuse to layer V of the cortex to directly depolarise the pyramidal neurones which form the corticostriatal pathway. This diffusion will take a certain amount of time, which may explain the
delay seen in the present experiments. The reason why both NMDA and potassium, when topically applied fail to depolarise immediately pyramidal neurones in layer V of the cortex remains unclear. One possibility is that both excitatory and inhibitory neurotransmitters are released with no net effect on layer V.
In conclusion, the most likely reason for the observed effects of the topically applied drugs is that they diffuse through the cortex and affect striatal glutamate and aspartate release when the cell bodies and or dendrites of the neurones in layer V of the cortex are directly depolarised.