Faster Serotonin Clearance in CA3 Region of Hippocampus and Antidepressant-like Effect of Decynium-22 in Juvenile Mice Are Putatively Linked to Increased Plasma Membrane Monoamine Transporter Function: Implications for Efficacy of Antidepressants in Juveniles.

Selective serotonin reuptake inhibitors (SSRIs) are less efficacious in treating depression in children than in adults. SSRIs block serotonin uptake via the high-affinity, low-capacity serotonin transporter. However, the low-affinity, high-capacity organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT) are emerging as important players in serotonin uptake. We hypothesized that OCT3 and/or PMAT are functionally upregulated in juveniles, thereby buffering SSRIs' ability to enhance serotonergic neurotransmission. Unlike in adult mice, we found the OCT/PMAT blocker, decynium-22, to have standalone antidepressant-like effects in juveniles. Using in vivo high-speed chronoamperometry, we found that juveniles clear serotonin from the CA3 region of the hippocampus ~2-fold faster than adult mice. Cell density did not differ between ages, suggesting that faster serotonin clearance in juveniles is unrelated to faster diffusion through the extracellular matrix. Western blot and immunohistochemistry showed that juvenile mice have modestly greater expression of PMAT than adults, whereas OCT3 expression in the CA3 region of the hippocampus was similar between ages. Together, these data suggest that faster serotonin clearance and antidepressant-like effects of decynium-22 in juvenile mice may be due to functionally upregulated PMAT. Faster serotonin clearance via PMAT in juveniles may contribute to reduced therapeutic efficacy of SSRIs in children relative to adults.

High Affinity Decynium-22 Binding to Brain Membrane Homogenates and Reduced Dorsal Camouflaging after Acute Exposure to it in Zebrafish.

Organic cation transporters (OCTs) are expressed in the mammalian brain, kidney, liver, placenta, and intestines, where they facilitate the transport of cations and other substrates between extracellular fluids and cells. Despite increasing reliance on ectothermic vertebrates as alternative toxicology models, properties of their OCT homologs transporting many drugs and toxins remain poorly characterized. Recently, in zebrafish (Danio rerio), two proteins with functional similarities to human OCTs were shown to be highly expressed in the liver, kidney, eye, and brain. This study is the first to characterize in vivo uptake to the brain and the high-affinity brain membrane binding of the mammalian OCT blocker 1-1'-diethyl-2,2'cyanine iodide (decynium-22 or D-22) in zebrafish. Membrane saturation binding of [3H] D-22 in pooled zebrafish whole brain versus mouse hippocampal homogenates revealed a high-affinity binding site with a KD of 5 ± 2.5 nM and Bmax of 1974 ± 410 fmol/mg protein in the zebrafish brain, and a KD of 3.3 ± 2.3 and Bmax of 704 ± 182 fmol/mg protein in mouse hippocampus. The binding of [3H] D-22 to brain membrane homogenates was partially blocked by the neurotoxic cation 1-methyl-4-phenylpyridinium (MPP+), a known OCT substrate. To determine if D-22 bath exposures reach the brain, zebrafish were exposed to 25 nM [3H] D-22 for 10 min, and 736 ± 68 ng/g wet weight [3H] D-22 was bound. Acute behavioral effects of D-22 in zebrafish were characterized in two anxiety-relevant tests. In the first cohort of zebrafish, 12.5, 25, or 50 mg/L D-22 had no effect on their height in the dive tank or entries and time spent in white arms of a light/dark plus maze. By contrast, 25 mg/L buspirone increased zebrafish dive tank top-dwelling (p < 0.05), an anticipated anxiolytic effect. However, a second cohort of zebrafish treated with 50 mg/L D-22 made more white arm entries, and females spent more time in white than controls. Based on these findings, it appears that D-22 bath treatments reach the zebrafish brain and have partial anxiolytic properties, reducing anti-predator dorsal camouflaging, without increasing vertical exploration. High-affinity binding of [3H] D-22 in zebrafish brain and mouse brain was similar, with nanomolar affinity, possibly at conserved OCT site(s).

Effect of concurrent organic cation transporter blockade on norepinephrine clearance inhibiting- and antidepressant-like actions of desipramine and venlafaxine.

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Studies have shown that blockade of low-affinity/high-capacity transporters, such as organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT), with decynium-22 can produce antidepressant-like effects and inhibit serotonin clearance in brain when the serotonin transporter is pharmacologically or genetically compromised. In vitro studies show that OCTs/PMAT are also capable of norepinephrine transport, raising the possibility that decynium-22 might enhance the antidepressant-like effects of norepinephrine transporter inhibitors. Using in vivo electrochemistry, we show that local administration of decynium-22 into dentate gyrus of hippocampus enhanced the ability of the norepinephrine transporter blocker, desipramine, but not the dual norepinephrine/serotonin transporter blocker venlafaxine, to inhibit norepinephrine clearance. In parallel, systemic administration of decynium-22 (0.32 mg/kg) enhanced the antidepressant-like effects of desipramine (32 mg/kg), but not those of venlafaxine, in the tail suspension test, underscoring the heterogeneous response of mice to antidepressants, including those that share similar mechanisms of action. Systemic administration of normetanephrine, a potent blocker of OCT3, failed to potentiate the antidepressant-like effects of desipramine, suggesting that the actions of decynium-22 to augment the antidepressant-like effects of desipramine are likely mediated by another OCT isoform and/or PMAT. Taken together with existing literature, concurrent blockade of OCTs and/or PMAT merits further investigation as an adjunctive therapeutic for desipramine-like antidepressant drugs.

Histamine Receptors Regulate the Activity, Surface Expression, and Phosphorylation of Serotonin Transporters.

Reuptake and clearance of released serotonin (5-HT) are critical in serotonergic neurotransmission. Serotonin transporter (SERT) is mainly responsible for clearing the extracellular 5-HT. Controlled trafficking, phosphorylation, and protein stability have been attributed to robust SERT activity. H3 histamine receptors (H3Rs) act in conjunction and regulate 5-HT release. H3Rs are expressed in the nervous system and located at the serotonergic terminals, where they act as heteroreceptors. Although histaminergic and serotonergic neurotransmissions are thought to be two separate events, whether H3Rs influence SERT in the CNS to control 5-HT reuptake has never been addressed. With a priori knowledge gained from our studies, we explored the possibility of using rat hippocampal synaptosomal preparations. We found that treatment with H3R/H4R-agonists immepip and (R)-(-)-α-methyl-histamine indeed resulted in a time- and concentration-dependent decrease in 5-HT transport. On the other hand, treatment with H3R/H4R-inverse agonist thioperamide caused a moderate increase in 5-HT uptake while blocking the inhibitory effect of H3R/H4R agonists. When investigated further, immepip treatment reduced the level of SERT on the plasma membrane and its phosphorylation. Likewise, CaMKII inhibitor KN93 or calcineurin inhibitor cyclosporine A also inhibited SERT function; however, an additive effect with immepip was not seen. High-speed in vivo chronoamperometry demonstrated that immepip delayed 5-HT clearance while thioperamide accelerated 5-HT clearance from the extracellular space. Immepip selectively inhibited SERT activity in the hippocampus and cortex but not in the striatum, midbrain, and brain stem. Thus, we report here a novel mechanism of regulating SERT activity by H3R-mediated CaMKII/calcineurin pathway in a brain-region-specific manner and perhaps synaptic 5-HT in the CNS that controls 5-HT clearance.

Eating high fat chow decreases dopamine clearance in adolescent and adult male rats but selectively enhances the locomotor stimulating effects of cocaine in adolescents.

BACKGROUND: Feeding conditions can influence dopamine neurotransmission and impact behavioral and neurochemical effects of drugs acting on dopamine systems. This study examined whether eating high fat chow alters the locomotor effects of cocaine and dopamine transporter activity in adolescent (postnatal day 25) and adult (postnatal day 75) male Sprague-Dawley rats. METHODS: Dose-response curves for cocaine-induced locomotor activity were generated in rats with free access to either standard or high fat chow or restricted access to high fat chow (body weight matched to rats eating standard chow). RESULTS: Compared with eating standard chow, eating high fat chow increased the sensitivity of adolescent, but not adult, rats to the acute effects of cocaine. When tested once per week, sensitization to the locomotor effects of cocaine was enhanced in adolescent rats eating high fat chow compared with adolescent rats eating standard chow. Sensitization to cocaine was not different among feeding conditions in adults. When adolescent rats that previously ate high fat chow ate standard chow, sensitivity to cocaine returned to normal. As measured by chronoamperometry, dopamine clearance rate in striatum was decreased in both adolescent and adult rats eating high fat chow compared with age-matched rats eating standard chow. CONCLUSIONS: These results suggest that high fat diet-induced reductions in dopamine clearance rate do not always correspond to increased sensitivity to the locomotor effects of cocaine, suggesting that mechanisms other than dopamine transporter might play a role. Moreover, in adolescent but not adult rats, eating high fat chow increases sensitivity to cocaine and enhances the sensitization that develops to cocaine.

5-HT1B receptor modulation of the serotonin transporter in vivo: studies using KO mice.

The serotonin transporter (SERT) controls the strength and duration of serotonergic neurotransmission by the high-affinity uptake of serotonin (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 [(3)H]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.

Decynium-22 enhances SSRI-induced antidepressant-like effects in mice: uncovering novel targets to treat depression.

Mood disorders cause much suffering and lost productivity worldwide, compounded by the fact that many patients are not effectively treated by currently available medications. The most commonly prescribed antidepressant drugs are the selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act by blocking the high-affinity 5-HT transporter (SERT). The increase in extracellular 5-HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low-affinity, high-capacity transporters for 5-HT in brain [i.e., organic cation transporters (OCTs) and plasma membrane monoamine transporter], which may limit the ability of SSRIs to increase extracellular 5-HT. Decynium-22 (D-22) is a blocker of these transporters, and using this compound we uncovered a significant role for OCTs in 5-HT uptake in mice genetically modified to have reduced or no SERT expression (Baganz et al., 2008). This raised the possibility that pharmacological inactivation of D-22-sensitive transporters might enhance the neurochemical and behavioral effects of SSRIs. Here we show that in wild-type mice D-22 enhances the effects of the SSRI fluvoxamine to inhibit 5-HT clearance and to produce antidepressant-like activity. This antidepressant-like activity of D-22 was attenuated in OCT3 KO mice, whereas the effect of D-22 to inhibit 5-HT clearance in the CA3 region of hippocampus persisted. Our findings point to OCT3, as well as other D-22-sensitive transporters, as novel targets for new antidepressant drugs with improved therapeutic potential.

Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter.

Serotonin [i.e., 5-hydroxytryptamine (5-HT)]-targeted antidepressants are in wide use for the treatment of mood disorders, although many patients do not show a response or experience unpleasant side effects. Psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (i.e., "ecstasy"), also impact 5-HT signaling. To help dissect the contribution of 5-HT signaling to the actions of these and other agents, we developed transgenic mice in which high-affinity recognition of multiple antidepressants and cocaine is eliminated. Our animals possess a modified copy of the 5-HT transporter (i.e., SERT, slc6a4) that bears a single amino acid substitution, I172M, proximal to the 5-HT binding site. Although the M172 substitution does not impact the recognition of 5-HT, this mutation disrupts high-affinity binding of many competitive antagonists in transfected cells. Here, we demonstrate that, in M172 knock-in mice, basal SERT protein levels, 5-HT transport rates, and 5-HT levels are normal. However, SERT M172 mice display a substantial loss of sensitivity to the selective 5-HT reuptake inhibitors fluoxetine and citalopram, as well as to cocaine. Through a series of biochemical, electrophysiological, and behavioral assays, we demonstrate the unique properties of this model and establish directly that SERT is the sole protein responsible for selective 5-HT reuptake inhibitor-mediated alterations in 5-HT clearance, in 5-HT1A autoreceptor modulation of raphe neuron firing, and in behaviors used to predict the utility of antidepressants.

Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonin-transporter-deficient mice.

Mood disorders cause much suffering and are the single greatest cause of lost productivity worldwide. Although multiple medications, along with behavioral therapies, have proven effective for some individuals, millions of people lack an effective therapeutic option. A common serotonin (5-HT) transporter (5-HTT/SERT, SLC6A4) polymorphism is believed to confer lower 5-HTT expression in vivo and elevates risk for multiple mood disorders including anxiety, alcoholism, and major depression. Importantly, this variant is also associated with reduced responsiveness to selective 5-HT reuptake inhibitor antidepressants. We hypothesized that a reduced antidepressant response in individuals with a constitutive reduction in 5-HTT expression could arise because of the compensatory expression of other genes that inactivate 5-HT in the brain. A functionally upregulated alternate transporter for 5-HT may prevent extracellular 5-HT from rising to levels sufficiently high enough to trigger the adaptive neurochemical events necessary for therapeutic benefit. Here we demonstrate that expression of the organic cation transporter type 3 (OCT3, SLC22A3), which also transports 5-HT, is upregulated in the brains of mice with constitutively reduced 5-HTT expression. Moreover, the OCT blocker decynium-22 diminishes 5-HT clearance and exerts antidepressant-like effects in these mice but not in WT animals. OCT3 may be an important transporter mediating serotonergic signaling when 5-HTT expression or function is compromised.