Clearance rates for serotonin (5-HT) in heterozygote (+/-) and homozygote (-/-) serotonin transporter (5-HTT) knockout (KO) mice have not been determined in vivo. Moreover, the effect of selective serotonin reuptake inhibitors (SSRIs) on 5-HT clearance in these mice has not been examined. In this study, the rate of clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus of anesthetized mice using high-speed chronoamperometry. Compared with wild-type mice, the maximal rate of 5-HT clearance from extracellular fluid (ECF) was decreased in heterozygotes and more markedly so in KO mice. Heterozygote mice were more sensitive to the 5-HT uptake inhibitor, fluvoxamine, resulting in longer clearance times for 5-HT than in wild-type mice; as expected, the KO mice were completely unresponsive to fluvoxamine. There were no associated changes in norepinephrine transporter density, nor was there an effect of the norepinephrine uptake inhibitor, desipramine, on 5-HT clearance in any genotype. Thus, adaptive changes in the norepinephrine transport system do not occur in the CA3 region of hippocampus as a consequence of 5-HTT KO. These data highlight the potential of the heterozygote 5-HTT mutant mice to model the dynamic in vivo consequences of the human 5-HTT polymorphism. Keywords: chronoamperometry, hippocampus, serotonin clearance rates, serotonin transporter, serotonin transporter knockout mice, norepinephrine transporter. The serotonin transporter (5-HTT) terminates serotonergic neurotransmission by high-affinity uptake of serotonin (5-HT) from extracellular fluid (ECF) and is a primary site of action for a number of psychotherapeutic drugs. To probe the involvement of the 5-HTT in mediating serotonergic neurotransmission Bengel et al. (1998) engineered mice with a targeted disruption of the 5-HTT gene, producing 5-HTT knockout (KO) mice. Studies characterizing the rate of 5-HT uptake into synaptosomes prepared from heterozygote (+/-) 5-HTT KO mice yielded unexpected results (Bengel et al. 1998). Although heterozygotes possess 50% fewer 5-HTTs than their wildtype (+/+) counterpart, rate of 5-HT uptake was not different between these genotypes. As expected, 5-HT uptake was non-existent in the null mutant (-/-). These data are in stark contrast to those reported for heterozygote dopamine (DA) transporter (DAT) KO mice where DA clearance is reduced by approximately 50%, commensurate with the reduction in transporter density (Jones et al. 1998).Differences in recording method may account, at least in part, for the unanticipated result in heterozygote 5-HTT KO mice. For example, in the study by Bengel and co-workers, uptake of [ 3 H]5-HT into synaptosomes was measured, whereas in studies in DAT KO mice, voltammetry was used to measure clearance of DA in brain slices (Jones et al. 1998). The primary difference between these methods is that the former measures uptake after an incubation period of several minutes, whereas voltammetric recordings permit

No abstract

No abstract

Termination of serotonergic transmission is the function of the plasma membrane 5-hydroxytryptamine (serotonin, 5-HT) transporter (SERT), which is also a high-affinity target in vivo for antidepressants, amphetamines, and cocaine. Studies show that SERT is regulated by protein kinase-and phosphataselinked pathways. In contrast, receptor-linked modulation of SERT is only minimally defined. Because noradrenergic stimulation is reported to influence 5-HT release, we explored possible presynaptic adrenoceptor-mediated regulation of SERT. In mouse forebrain synaptosomes, ␣ 2 -adrenoceptor agonists, particularly 5-bromo-N-[4,5-dihydro-1H-imidazol-2-yl]-6-quinoxalinamine (UK14304), triggered a concentration-and timedependent decrease in 5-HT transport. In contrast, 5-HT uptake was unaffected by pharmacological ␣ 1 -adrenoceptor activation. Kinetically, UK14304 significantly decreased the apparent substrate affinity, K m without altering transport capacity, V max . At concentrations of UK14304 supporting maximal inhibition of SERT in synaptosomes, no effect on SERT in transfected cells was observed, suggesting that UK14304 acts indirectly to reduce SERT activity. The effect of UK14304 on 5-HT uptake was not shared by other Na ϩ and Cl Ϫ -dependent transporters. UK14304-mediated inhibition of SERT function was yohimbine-sensitive, as was inhibition triggered by norepinephrine, and was abolished in the absence of added Ca 2ϩ . Moreover, UK14304 effects were attenuated by voltage-sensitive Ca 2ϩ channel antagonists, consistent with a role for Ca 2ϩ in UK14304 effects. In agreement with altered 5-HT transport activity in vitro, in vivo chronoamperometry studies revealed that UK14304 significantly prolonged 5-HT clearance. Our findings suggest that UK14304 modulates SERT function in vitro and in vivo via signaling pathways, possibly supported by an influx of Ca 2ϩ through voltage-sensitive Ca 2ϩ channels.It is well established that perturbation in monoaminergic transmission is a contributing factor in depression (Malison et al., 1998;Frazer, 2000). Consistent with these findings, the therapeutic actions of the first generation of antidepressants, the tricyclics, most likely exert their therapeutic actions by blocking the neuronal uptake of 5-hydroxytryptamine (serotonin, 5-HT) and/or norepinephrine (NE) (Barker and Blakely, 1995). In the central nervous system (CNS), 5-HT, and NE systems are themselves intimately connected. Electron microscopic autoradiography studies have provided evidence that serotonergic neurons in the rat dorsal raphe receive direct input from noradrenergic neurons (Baraban and Aghajanian, 1981). In addition, in vivo microdialysis studies (Numazawa et al., 1995;Gobert et al., 1998) and release experiments in brain slices (Scheibner et al., 2001) and synaptosomes (Maura et al., 1992;Gobbi et al., 1993a) have demonstrated that 5-HT release is modulated by the activation of presynaptic ␣ 2 -adrenoceptors. Such an interaction suggests a functional cross talk between the noradrenergic and the seroton...

No abstract

The serotonin transporter (SERT) controls the strength and duration of serotonergic neurotransmission by the high-affinity uptake of ser otonin (5-HT) from extracellular fluid. SERT is a key target for many psychotherapeutic and abused drugs, therefore understanding how SERT activity and expression are regulated is of fundamental importance. A growing literature suggests that SERT activity is under regulatory control of the 5-HT1B autoreceptor. The present studies made use of mice with a constitutive reduction (5-HT1B+/−) or knockout of 5-HT1B receptors (5-HT1B−/−), as well as mice with a constitutive knockout of SERT (SERT−/−) to further explore the relationship between SERT activity and 5-HT1B receptor expression. High-speed chronoamperometry was used to measure clearance of 5-HT from CA3 region of hippocampus in vivo. Serotonin clearance rate, over a range of 5-HT concentrations, did not differ among 5-HT1B receptor genotypes, nor did [3H]cyanoimipramine binding to SERT in this brain region, suggesting that SERT activity is not affected by constitutive reduction or loss of 5-HT1B receptors; alternatively, it might be that other transport mechanisms for 5-HT compensate for loss of 5-HT1B receptors. Consistent with previous reports, we found that the 5-HT1B receptor antagonist, cyanopindolol, inhibited 5-HT clearance in wild-type mice. However, this effect of cyanopindolol was lost in 5-HT1B−/− mice and diminished in 5-HT1B+/− mice, indicating that the 5-HT1B receptor is necessary for cyanopindolol to inhibit 5-HT clearance. Likewise, cyanopindolol was without effect on 5-HT clearance in SERT−/− mice, demonstrating a requirement for the presence of both SERT and 5-HT1B receptors in order for cyanopindolol to inhibit 5-HT clearance in CA3 region of hippocampus. Our findings are consistent with SERT being under the regulatory control of 5-HT1B autoreceptors. Future studies to identify signaling pathways involved may help elucidate novel therapeutic targets for the treatment of psychiatric disorders, particularly those linked to gene variants of the 5-HT1B receptor.

No abstract

The degree of occupancy of the serotonin transporter (SERT) by selective serotonin reuptake inhibitors (SSRIs) appears to be critical in determining therapeutic response. To gain insight into the extent of occupancy required to alter serotonergic neurotransmission we used high-speed chronoamperometry to determine the extent of serotonergic destruction required to reduce the clearance of exogenously administered serotonin from extracellular fluid in the CA3 region of the hippocampus. Rats were pretreated with various doses of 5,7-dihydroxytryptamine to produce either a low, intermediate or high loss of SERTs. Clearance of 5-HT was reduced only in rats with > 90% loss of SERT. In these rats, there was also a trend for peak signal amplitudes to be greater. There was no significant difference in these parameters between the sham group and those with low or intermediate loss of SERTs. The SSRI, fluvoxamine, prolonged clearance of 5-HT in sham, low and intermediate groups, whereas there was no effect of fluvoxamine in those rats with > 90% loss of SERT. Functional loss of SERT activity occurs when destruction of serotonergic innervation is greater than 90% but serotonin clearance and efficacy of fluvoxamine is maintained with as few as one fifth of a full complement of SERTs.