In this study, dietary n-3 PUFA supplementation to SHRs was associated with two distinct types of changes in ADHD-like behaviours which occured in synchrony with changes in neurotransmitter systems. One of these included reduced levels of reinforcer-controlled activity, impulsiveness and inattention in male SHR, with no or opposite effects in the female SHRs. Neurotransmitter dynamics in neostriatum of these animals were also changed, showing enhanced DA and 5-HT turnover, which again were predominantly seen in male SHRs. In contrast, the other change included a reduction in general locomotor activity in both SHR sexes, which appeared to correlate with changes in levels of several neuroactive amino acids in both male and female SHRs. Thus, our data indicate major sex-specific effects on neurotransmitter systems in the PUFA-fed animals, with the males having unique enhancements of both DA and 5-HT in the neostriatum. Although the 5-HT degradation product 5-HIAA also was significantly increased in females, with a similar turnover ratio to that seen in males, the turnover in female SHRs was not statistically significant. Even though the mechanisms behind these effects of n-3 PUFAs are unclear[9, 27, 54, 55], our data correlate well with the behavioural observations, which also showed sex differences, with males improving on all operant measures, whereas females did not. This similarity suggests a possible causal relation between the n-3 PUFA-induced increase of modulatory neurotransmission and improved behaviour.
Reinforcement mechanisms in the CNS are mostly mediated via DA synapses[57–59], which are heavily concentrated in neostriatal brain regions, particularly in the dorsal and ventral caudate-putamen. Here, excitatory Glu inputs from cerebral cortex and thalamus predominantly terminate on efferent GABAergic cells, with the widely distributed DA and 5-HT modulatory synaptic inputs from the brain stem having the ability to modulate the efficacy of the Glu-to-GABA synapses, thereby determining to what extent efferent GABAergic information will be transmitted out of the basal ganglia. This synaptic effect, mediated trough an undefined mechanism being involved in the neurobiological changes occurring in the n-3 PUFA-fed animals, may suggest enhanced action potential firing frequencies, structural synaptic changes (e.g. increased number of vesicles or synapses) or enhanced biochemical capacity, vesicle storage capacity or enhanced neurotransmitter levels[62–66].
In contrast, effects on locomotion appear to represent a distinct target, with general, non-operant locomotion activities being sensitive to n-3 PUFAs in both sexes. Given that the locomotion occurred continuously during the operant test, and that frame-to-frame analyses of video-recordings detected changes in pixels whenever the animals moved, it is clear that this measure reflected both operant behaviour controlled by the scheduled reinforcers as well as general movements. The interpretation that operant behaviour and video-measured locomotion represent two distinct phenomena is also supported by the dissociation of reinforcement-controlled lever pressing and changes in the video-measured locomotion observed in female SHRs, where n-3 PUFA feeding increased levels of operant responses but reduced initial video-measured locomotion.
Because the PUFA-induced reduction in non-operant behaviour locomotor activity occurred in both sexes, it is of great interest that those neuroactive transmitter amino acids which responded to n-3 PUFAs also showed similar patterns in the two sexes. Specifically, these included reduced plasma levels of Glu and enhanced levels of Gly, with no changes in Gln and GABA. Because the Gln/Glu ratio was significantly increased, glutamatergic neurotransmission in neostriatum of the SHRs may be enhanced by dietary n-3 PUFA supplementation in both sexes. Previous analysis has suggested that motor activity may be modulated by Glu receptor activation, in agreement with our observations.
The levels of Gly showed similar differences as those found in the Gln/Glu ratios; with low levels in the control-fed SHRs, and PUFA-induced increase in levels seen in both SHR sexes. Although Gly receptors are present in the rat striatum, and are proposed to modulate both cholinergic, dopaminergic and glutamatergic transmission[67–69], the importance of increased Gly levels in the striata of n-3 PUFA supplemented SHRs remains unknown[70–73]. Previous work has also indicated changes in Tau levels in male SHRs when compared to WKY controls, suggesting a possible role for Tau in association with ADHD. Indeed, Tau changes may lead to impaired regulation of Gly, GABA and/or DA signalling[52, 75–78]. However, Tau levels were only insignificantly reduced following n-3 PUFA feeding, making it unlikely that these changes represent an important mechanism for mediating n-3 PUFA-induced amelioration.
Finally, the PUFA-induced normalization of the Gln/Glu ratios as well as Gly levels, which occur in both female and male SHRs, is of considerable interest. This contrasts to the major PUFA-induced changes found in the DA and 5-HT turnover, seen predominantly in the male SHRs. Intuitively, these dietary effects of n-3 PUFA in ADHD models like SHR would be expected to normalize both groups of neurotransmitters. However, given the well-known distinct functional organizations of the quantitatively predominant amino acidergic synapses in the neostriatum as compared to the minor but widespread modulatory monoaminergic synaptic systems, our data appear consistent with the interpretation that n-3 PUFA feeding induced strong activation of the modulatory DA and 5-HT systems in order to normalize the functionally more important amino acidergic neurotransmitter systems. It should also be emphasized that our study only represents a small number of possible factors involved in ADHD-development. Moreover, other studies have also observed sex differences in other important neurochemical parameters like the dopamine D1 and D2 receptors, or monoamine-catabolizing enzymes like COMT[81, 82]. Finally, a potential weakness of our study is that the control group is fed considerably less fat than the n-3 PUFA-fed group. However, recent studies demonstrated that adult male Long Evans rats given a high-fat diet developed a decrease, not an increase in their DA turnover in the ventral striatum, making it highly unlikely that the high-fat diet per se employed in this study is responsible for the enhancement of DA turnover which occures in the male SHRs.