When three AD drugs with different pharmacologic actions – a tricylic (DMI) which primarily blocks norepinephrine reuptake, an SSRI (PAR) which primarily blocks serotonin reuptake, and a monoamine oxidase inhibitor (PHE) which retards the catabolism of all monoamines – were administered to rats for two weeks, certain changes in mRNA for TH, GAL, and GAL receptors were observed in different brain regions. Although the doses of these drugs that were used were standard in rat studies, the blood levels that were measured in this study were higher than normally encountered in therapeutic ranges, probably due to our having used fully mature male rats. However, dose-response analyses of several AD drugs that were carried out in our laboratory measuring effects on LC electrophysiological activity have shown that high blood levels of AD drugs are associated with similar effects on LC activity (i.e., inhibition of LC activity) to what is seen at lower therapeutic levels except that the change may be somewhat larger (West et al., 2009
); therefore, effects on mRNA reported here should be representative of what the drugs will produce at lower blood levels.
Salient changes were seen in the brain area that was the principal focus of this study, the LC. In LC cells, chronic administration of each of the three AD drugs significantly reduced GAL mRNA. The mRNA for TH in LC also was reduced by the three drugs; this effect was significant when the three ADs were considered together and compared with the vehicle-infused group. A similar finding in regard to the influence of chronic administration of AD drugs on the mRNA for TH in LC was previously reported in a well-known study by Nestler and colleagues (Nestler et al., 1990
). The findings reported here are consistent with the known effects of chronic administration of AD drugs on activity of LC neurons, which is to decrease their activity (for summary, see West et al., 2009
). Insofar as both TH and GAL are activity-dependent peptides – that is, their synthesis increases when cell bodies in which they are synthesized are active, and their synthesis decreases when these cell bodies are inactive – it would be expected that the inhibitory influence of AD drugs on the activity of LC neurons might well be reflected in decreased synthesis, reflected in decreased mRNA, for TH and GAL in these cells. That synthesis of both TH and GAL in LC is activity-dependent is supported by the finding that the mRNA for TH and for GAL in LC was highly correlated, as shown in . The correlations carried out used all of the animals in the study, thereby including vehicle-infused rats whose LC activity would have been higher than that of the animals infused with AD drugs; thus, the correlation shows, appropriately, a relationship seen across differing levels of LC activity. However, as also can be seen in , the positive relationship between TH and GAL mRNA was evident within each of the four groups in the study.
In the VTA, both GAL and TH mRNA showed the tendency to be reduced by AD treatment; these effects, however, neither of these effects reached two-tailed statistical significance. Nevertheless, such findings do not offer support for the theoretical interpretation presented in the introduction. This formulation proposed that effective AD treatment will, by decreasing LC activity, decrease release of GAL in VTA, thereby increasing VTA-DA neuronal activity. The findings reported here point to the opposite effect. When Razani et al. (2000)
examined changes in DRN produced by infusion of GAL into the ventricular system of the rat brain, they found that this infusion decreased GAL mRNA in DRN. Such findings indicate that increased extracellular levels of GAL (via the infusion) caused a decrease in GAL mRNA in the cell bodies of DRN that presumably were affected by the GAL infusion. While the Razani et al study did not assess changes in VTA, the findings would seem applicable to VTA-DA neurons in that extracellular GAL exerts an inhibitory (hyperpolarizing) influence on both VTA-DA and DRN neurons (refs). Thus, decreased mRNA for both TH and GAL mRNA in VTA resulting from AD treatment suggests that AD treatment may have decreased VTA-DA activity, perhaps by increasing extracellular GAL in VTA or by some other mechanism. However, this conclusion is tempered by the fact that DA neurons in the VTA region represent a relatively small number interdigitated amongst other neurons that synthesize different transmitters, and, consequently, it is unclear as to which neurons contributed to the mRNA measured in the VTA sample. While the TH-containing neurons in VTA are catecholaminergic, the same cannot be said for GAL. Without in situ determination, which was not performed, a firm conclusion as to what neurons in this region contributed to GAL mRNA is not possible.
The largest change in mRNA seen in the VTA region was a decrease in mRNA for GalR2 receptors, which is particularly interesting in view of the report by Kuteeva et al. (2008)
suggesting that stimulation of GalR2 receptors mediates antidepressant action. If this is correct, decreased synthesis of Gal R2 receptors in the VTA region could point to antidepressant activity taking place, with the decrease in mRNA for this receptor resulting either from negative feedback on synthesis of this receptor because such action is occurring or from direct stimulation of GalR2 receptors by GAL to bring about an antidepressant effect.
The other two brains regions examined were A1/C1 and DRN. In the A1/C1 region, AD treatment showed a tendency to decrease TH and GAL mRNA, so that this region looked similar to LC and VTA in this regard, but this effect in the A1/C1 region did not reach statistical significance. In the DRN, however, a markedly different pattern for GAL mRNA was seen in relation to the AD treatment than was evident in the other three brain regions. GAL mRNA, which was also the only mRNA in DRN to be significantly affected by different drug treatments, was markedly increased in the animals that had received PAR. This increase was distinctly different from what was seen in all other groups, and significantly different from VEH- and DMI-treated animals. The study by Razani et al. (2000)
clearly demonstrates an interaction between GAL and 5-HT1A receptors in DRN neurons, suggesting that action of 5-HT and GAL is linked in these cells. The results shown here reinforce this interaction, and, moreover, indicate that activation of 5-HT1A receptors, as will occur with PAR treatment, profoundly affects GAL mRNA in DRN neurons. Additionally, insofar as the effect of PAR treatment on GAL mRNA in DRN differed for that of DMI treatment, the results presented here point to an effect of a 5-HT-reuptake-blocking drug on GAL that is not shared by ADs that do not possess this capacity.
An interesting aspect of the present findings is the ability to examine intercorrelations of the various mRNAs within brain regions. As described earlier, TH and GAL mRNA were highly correlated in the LC, consistent with the idea that (1) these peptides are found within the same cells in this region/sample/(presumably LC neurons), and (2) the mRNAs are highly correlated because the two peptides are both activity dependent. Similarly, in the A1/C1 and VTA regions, although colocalization of GAL and TH in catecholaminergic neurons in these brain regions is much less extensive or evident than in LC (e.g., Levin et al., 1987
; Skofitsch and Jacobowitz, 1985
), TH and GAL mRNA was also significantly correlated. In contrast, in the DRN region GAL and TH mRNA were not correlated (in fact, were somewhat negatively correlated). In DRN, unlike the other brain regions examined in this study, the mRNA for TH and much of the mRNA for GAL is clearly localized in different neurons in the sample – TH would be found in a small number of dopaminergic cells in this region designated as A10c and A10dc (Hökfelt et al., 1984
) whereas much of the GAL mRNA would be colocalized in the more numerous serotonergic cells in DRN (e.g., Melander et al., 1986
; Xu and Hökfelt, 1997
); thus, it is perhaps not surprising that the DRN does not show the same relationship between mRNA for GAL and TH evident in the LC and in the two other catecholaminergic regions.
Also of interest, correlations between mRNAs for the three types of GAL receptors (GalR1, GalR2, and GalR3) indicated that these were associated differently in the brain regions examined. In the catecholaminergic cell-body regions (LC, A1/C1, and VTA), the mRNA for GalR2 and GalR3 was significantly correlated (r=.62, .78, and .41 respectively) whereas mRNA for GalR1 did not correlate with either of these. In contrast to this, in DRN GalR1 mRNA correlated highly with GalR3 mRNA (r=.74). This suggests that synthesis of GalR2 receptors is associated, or co-varies, with synthesis of GalR3 receptors in the catecholaminergic cell-body regions studied, whereas synthesis of GalR1 receptors is strongly associated, or co-varies, with synthesis of GalR3 receptors in the DRN region.
In summary, chronic administration of AD drugs that represented three classes of such drugs (i.e, tricyclic, SSRI, and MAOI) affected mRNA for GAL, GAL receptors, and TH in four brain regions studied. In the cell bodies of LC neurons, GAL and TH mRNA were decreased; insofar as GAL and TH are activity-dependent peptides, this effect is consistent with decreases in LC activity that are produced by chronic administration of these AD drugs. In the A1/C1 region and in VTA, GAL and TH mRNA also tended to be decreased. In VTA, the most marked change in GAL receptor mRNA produced by AD administration was a decrease in mRNA for GAL2 receptors. In DRN, the pattern of mRNA changes differed from that seen in the other brain regions studied, both in effects on GAL mRNA and with regard to intercorrelations of GAL receptor mRNA changes. These results confirm that, in brain regions that appear to play an important role in depression, mRNA for GAL, its receptors, and TH are altered with chronic administration of AD drugs that produces therapeutic effects of these drugs.