An in vitro superfusion slice technique was used to measure simultaneous release of [3H]DA and [14C]acetylcholine from pre-labeled cross-chopped slices of nucleus accumbens or caudate-putamen (20-22). Neurotransmitter release was evoked either by electrical stimulation (2 minute trains of biphasic square wave pulses each of 2 ms duration, 16 mA amplitude and 5 Hz frequency) or by exposure of the slices to a high extracellular K+ (25 mM) concentration, or to drugs such as d-amphetamine or methylphenidate. Exposure to 25 mM K+ caused ten-times greater release of [3H]DA from nucleus accumbens and caudate-putamen slices than electrical stimulation.

Results show that depolarization(25 mM K+)-induced release of DA from nucleus accumbens slices of SHR was significantly lower than that of their Wistar-Kyoto control rats (WKY) (23). This difference was not observed when neurotransmitter release was evoked by electrical stimulation of the slices presumably because electrical stimulation released ten times less DA than K+ stimulation (22,24). The increased demand on tissue DA reserves might have been required to reveal the difference between SHR and WKY nucleus accumbens slices. This is in contrast to the caudate-putamen where both 25 mM K+ and electrically stimulated release of DA was significantly lower in SHR than WKY (22,23,25).

The in vitro superfusion slice technique provides an opportunity to study neurotransmitter receptor function. DA autoreceptor mediated inhibition of electrically stimulated DA release was significantly greater in caudate-putamen but not nucleus accumbens slices of SHR compared to WKY possibly accounting for the decreased release of DA observed in SHR caudate-putamen slices (22,25). Evidence was however obtained to suggest that DA autoreceptor efficacy was increased in nucleus accumbens tissue at low endogenous agonist concentrations (22). D2-like receptor blockade by the D2 antagonist, sulpiride, caused a significantly greater increase in the electrically stimulated release of DA from nucleus accumbens slices of SHR compared to WKY. A similar result was obtained when 25 mM K+ was used to stimulate DA release, sulpiride caused significantly greater release of [3H]DA from SHR nucleus accumbens slices than from WKY slices (unpublished data). Activation of DA autoreceptors by quinpirole produced a similar dose-dependent inhibition of electrically stimulated DA release in SHR and WKY nucleus accumbens slices, suggesting that the difference in autoreceptor function might signify a difference in efficacy of endogenous DA rather than a change in SHR DA autoreceptor number (22). When DA autoreceptors were antagonized, SHR nucleus accumbens slices released 10% more [3H]DA than WKY, suggesting increased autoreceptor mediated inhibition of DA release in SHR. This could have occurred as a result of abnormally elevated synaptic DA concentrations at an early stage of development.

Subsequent in vitro superfusion studies revealed that methylphenidate, a drug frequently used in the treatment of ADHD, released significantly less DA from nucleus accumbens slices of SHR than from WKY, while d-amphetamine, in contrast to methylphenidate, released more DA from nucleus accumbens and caudate-putamen slices of SHR compared to WKY (26). d-Amphetamine enters DA terminals and vesicles and causes DA release by means of the DA uptake carrier (27). Although SHR have been reported to have an increased number of DA transporters (28), the DA uptake carrier appeared to be functioning normally in both nucleus accumbens and caudate-putamen slices of SHR. Inhibition of uptake by low concentrations of an uptake blocker increased the electrically stimulated release of DA to the same extent in SHR and WKY (25,26). The results suggest that vesicle storage of DA might be impaired in SHR, causing leakage of DA into the cytoplasm, since SHR released less DA from vesicle stores in response to methylphenidate (nucleus accumbens), 25 mM K+ (nucleus accumbens and caudate-putamen) or electrical (caudate-putamen) stimulation and released more DA from cytoplasmic stores via the uptake carrier in response to d-amphetamine. This is consistent with the finding that redistribution of DA from vesicles to the cytoplasm by the use of a reserpine-like compound, Ro4-1284, did not increase extracellular DA in striatal slices of intact mice and that subsequent addition of d-amphetamine was required to induce rapid release of DA (27). SHR appear to have raised cytoplasmic concentrations of DA which may be due to leakage from the vesicles which was revealed by addition of d-amphetamine to the slices (26).

Altogether, these findings suggest that presynaptic regulation of DA release has been altered in SHR to cause down-regulation of the DA system, which is reflected in a decreased response of DA terminals to depolarization-induced release of DA from vesicle stores. Both nucleus accumbens and caudate-putamen are affected, the difference is not confined to the nucleus accumbens. It is a more generalized effect. However, it does not extend to all neurotransmitters. There was no evidence of impairment of acetylcholine release from nucleus accumbens or caudate-putamen slices (22). However, if the DA vesicle transporter is at fault then other monoamine neurotransmitters may also be affected. There is evidence from HPLC studies that norepinephrine and serotonin may be affected in SHR (24,29).

The down-regulation of SHR DA transmission could have occurred at an early stage of development when D4 and D2 receptor levels were still high (30). Elimination of excessive amounts of DA receptors which normally occurs during maturation (30), might not have occurred to the same extent in SHR as in WKY, especially as far as caudate-putamen DA autoreceptors are concerned. The alteration of DA release mechanisms in the nucleus accumbens appears to be more subtle than in the caudate-putamen, with evidence suggesting increased efficacy of DA autoreceptors at endogenous DA levels, perhaps involving altered coupling to second messenger systems or their intracellular effectors.

The impairment in DA transmission could have left the adult SHR with compromised DA reward/reinforcement mechanisms resulting in the behavioral disturbances characteristic of ADHD. The very necessary DA-mediated facilitation of integration of afferent signals in the nucleus accumbens could be impaired. DA neurons respond phasically to alerting stimuli. (6) If stimulus-evoked release of DA were deficient, the required postsynaptic effects resulting from activation of D1-like receptors e.g. activation of CREB, would be impaired. Indeed, consistent with the proposed deficiency in stimulation-evoked DA release, is the finding that postsynaptic D1-like receptors are up-regulated and expression of transcription factor genes, c-fos and zif-268, is reduced in the brains of SHR (28,31,32).

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