MAB1564 Sigma-AldrichAnti-Serotonin Transporter Antibody, clone 17-7A4

Major depressive disorder (MDD) is a prevalent mood disorder that is associated with differential prefrontal brain expression patterns. Treatment of MDD includes a variety of biopsychosocial approaches. In medical practice, antidepressant drugs are the most common treatment for depressive episodes, and they are among the most prescribed medications in North America. Although antidepressants are clearly effective, particularly for moderate to severe depressive episodes, there is variability in how individuals respond to antidepressant treatment. Failure to respond has individual, economic and social consequences for patients and their families. Several lines of evidence demonstrate that genes are regulated through the activity of microRNAs (miRNAs), which act as fine-tuners and on-off switches of gene expression. Here we report on complementary studies using postmortem human brain samples, cellular assays and samples from clinical trials of patients with depression and show that miR-1202, a miRNA specific to primates and enriched in the human brain, is differentially expressed in individuals with depression. Additionally, miR-1202 regulates expression of the gene encoding metabotropic glutamate receptor-4 (GRM4) and predicts antidepressant response at baseline. These results suggest that miR-1202 is associated with the pathophysiology of depression and is a potential target for new antidepressant treatments.

The present study investigated whether the rotation rate induced by amphetamine in 6-OHDA-lesioned rats was predictive of development of L-DOPA-induced dyskinesia (LID) and success of the lesion procedure in our experimental settings. We collected data from 312 6-OHDA-lesioned rats (from different sets of experiments). Rats were subjected to the amphetamine-induced rotation test (2.5mg/kg) and chronically treated with L-DOPA (6 mg/kg) to establish dyskinesia. A poor correlation was present between amphetamine-induced rotation and LID. Moreover, no correlation was found between amphetamine-induced rotation and tyrosine hydroxylase (TH) positive cell number in the lesioned substantia nigra pars compacta, while there was a weak correlation between the percentage of TH positive cell number and LID. These results indicate that the amphetamine-induced rotation test is a poor predictor of the 6-OHDA-lesion success, as well as of the development of LID at the dose of amphetamine used here. Our data also suggest that all rats with amphetamine-induced rotation ≥ 3 turns/min should be included in dyskinesia studies, as they showed the same propensity to develop dyskinesia. Moreover, SERT expression levels suggest that reduced striatal and pallidal serotonin innervation might have contributed to the lower dyskinesia levels observed in a subset of amphetamine-responsive rats.

Dyskinesia seen in the off-state, referred as graft-induced dyskinesia (GID), has emerged as a serious complication induced by dopamine (DA) cell transplantation in parkinsonian patients. Although the mechanism underlying the appearance of GID is unknown, in a recent clinical study the partial 5-HT(1A) agonist buspirone was found to markedly reduce GID in three grafted patients, who showed significant serotonin (5-HT) hyperinnervation in the grafted striatum in positron emission tomography scanning (Politis et al., 2010, 2011). Prompted by these findings, this study was performed to investigate the involvement of serotonin neurons in the appearance of GID in the rat 6-hydroxydopamine model. L-DOPA-primed rats received transplants of DA neurons only, DA plus 5-HT neurons or 5-HT neurons only into the lesioned striatum. In DA cell-grafted rats, with or without 5-HT neurons, but not in 5-HT grafts, GID was observed consistently after administration of amphetamine (1.5mg/kg, i.p.) indicating that grafted DA neurons are required to induce GID. Strikingly, a low dose of buspirone produced a complete suppression of GID. In addition, activation of 5-HT(1A) and 5-HT(1B) receptors by 8-OH-DPAT and CP 94253, known to inhibit the activity of 5-HT neurons, significantly reduced GID, whereas induction of neurotransmitter release by fenfluramine administration significantly increased GID, indicating an involvement of the 5-HT system in the modulation of GID. To investigate the involvement of the host 5-HT system in GID, the endogenous 5-HT terminals were removed by intracerebral injection of 5,7-dihydroxytryptamine, but this treatment did not affect GID expression. However, 5-HT terminal destruction suppressed the anti-GID effect of 5-HT(1A) and 5-HT(1B) agonists, demonstrating that the 5-HT(1) agonist combination exerted its anti-GID effect through the activation of pre-synaptic host-derived receptors. By contrast, removal of the host 5-HT innervation or pre-treatment with a 5-HT(1A) antagonist did not abolish the anti-GID effect of buspirone, showing that its effect is independent from activation of either pre- or post-synaptic 5-HT(1A) receptors. Since buspirone is known to also act as a DA D(2) receptor antagonist, the selective D(2) receptor antagonist eticlopride was administered to test whether blockade of D(2) receptors could account for the anti-dyskinetic effect of buspirone. In fact, eticlopride produced complete suppression of GID in grafted animals already at very low dose. Together, these results point to a critical role of both 5-HT(1) and D(2) receptors in the modulation of GID, and suggest that 5-HT neurons exert a modulatory role in the development of this side effect of neuronal transplantation.

Several lines of evidence have implicated a direct reciprocal interaction between serotonin and nitric oxide (NO). The goal of this investigation was, therefore, to examine the coexpression of tryptophan hydroxylase (TPH; the rate limiting enzyme for the synthesis of serotonin) and neuronal NO synthase (nNOS) in the ascending cortical projecting raphe nuclei (B6-B9 subgroups), when compared with the descending spinal cord projecting raphe nuclei (B1-B3 subgroups). Our data demonstrated that: (1) a significant number of raphe-cortical projecting neurons was identified not only in the midline subgroup of dorsal raphe (B6, 7) but also in the median raphe (B8), as well as in the supralemniscal nucleus (B9); (2) serotonergic cortical projecting neurons from these three raphe nuclei exhibited a high (>80%) percentage of coexpression with nNOS immunoreactivity; (3) similarly, serotonin transporter immunoreactive fibers in the medial prefrontal cortex were also double-labeled with nNOS immunoreactivity; (4) in contrast, the descending spinal cord projecting raphe nuclei revealed only TPH but not nNOS immunoreactivity. Our present findings suggest the existence of a direct interaction between serotonin and NO in the ascending cortical projecting raphe system. In contrast, a different strategy appears to operate the descending spinal cord projecting raphe system.

Previously, we found that the brainstem neuronal network in normal rats undergoes abrupt neurochemical, metabolic, and physiological changes around postnatal days (P) 12-13, a critical period when the animal's response to hypoxia is also the weakest. This has special implications for sudden infant death syndrome (SIDS), insofar as seemingly normal infants succumb to SIDS when exposed to respiratory stressors (e.g., hypoxia) during a narrow postnatal window. Because an abnormal serotonergic system has recently been implicated in SIDS, we conducted a large-scale investigation of the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) and serotonin transporter (SERT) with semiquantitative immunohistochemistry in multiple brainstem nuclei of normal rats aged P2-21. We found that 1) TPH and SERT immunoreactivity in neurons of raphé magnus, obscurus, and pallidus and SERT in the neuropil of the pre-Bötzinger complex, nucleus ambiguus, and retrotrapezoid nucleus were high at P2-11 but decreased markedly at P12 and plateaued thereafter until P21; 2) SERT labeling in neurons of the lateral paragigantocellular nucleus (LPGi) and parapyramidal region (pPy) was high at P2-9 but fell significantly at P10, followed by a gradual decline until P21; 3) TPH labeling in neurons of the ventrolateral medullary surface was stable except for a significant fall at P12; and 4) TPH and SERT immunoreactivity in a number of other nuclei was relatively stable from P2 to P21. Thus, multiple brainstem nuclei exhibited a significant decline in TPH and SERT immunoreactivity during the critical period, suggesting that such normal development can contribute to a narrow window of vulnerability in postnatal animals.

Previous studies have shown that serotonin neurons play an important role in the induction and maintenance of L-DOPA-induced dyskinesia in animals with lesion of the nigrostriatal dopamine system. Patients with Parkinson's disease that receive transplants of foetal ventral mesencephalic tissue, the graft cell preparation is likely to contain, in addition to dopamine neurons, serotonin neurons that will vary in number depending on the landmarks used for dissection. Here, we have studied the impact of grafted serotonin neurons--alone or mixed with dopamine neurons--on the development of L-DOPA-induced dyskinesia in rats with a partial 6-hydroxydopamine lesion of the host nigrostriatal projection. In these rats, which showed only low-level dyskinesia at the time of transplantation, serotonin grafts induced a worsening in the severity of dyskinesia that developed during continued L-DOPA treatment, while the dopamine-rich graft had the opposite, dampening effect. The detrimental effect seen in animals with serotonin neuron grafts was dramatically increased when the residual dopamine innervation in the striatum was removed by a second 6-hydroxydopamine lesion. Interestingly, rats with grafts that contained a mixture of dopamine and serotonin neurons (in approximately 2:1) showed a marked reduction in L-DOPA-induced dyskinesia over time, and the appearance of severe dyskinesia induced by the removal of the residual dopamine innervation, seen in the animals with transplants of serotonin neurons alone, was blocked. FosB expression in the striatal projection neurons, which is associated with dyskinesias, was also normalized by the dopamine-rich grafts, but not by the serotonin neuron grafts. These data indicate that as long as a sufficient portion, some 10-20%, of the dopamine innervation still remains, the increased host serotonin innervation generated by the grafted serotonin neurons will have limited effect on the development or severity of L-DOPA-induced dyskinesias. At more advanced stages of the disease, when the dopamine innervation of the putamen is reduced below this critical threshold, grafted serotonin neurons are likely to aggravate l-DOPA-induced dyskinesia in those cases where the dopamine re-innervation derived from the grafted neurons is insufficient in magnitude or do not cover the critical dyskinesia-inducing sub-regions of the grafted putamen. We conclude that it is not the absolute number of serotonin neurons in the grafts, but the relative densities of dopamine and serotonin innervations in the grafted striatum that is the critical factor in determining the long-term effect of foetal tissue graft, beneficial or detrimental, on dyskinesia in grafted Parkinson's disease patients.

Clinical trials in patients with Parkinson's disease have shown that transplants of fetal mesencephalic dopamine neurons can form a new functional innervation of the host striatum, but the clinical benefits have been highly variable: some patients have shown substantial recovery in motor function, whereas others have shown no improvement and even a worsening in the 3,4-dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinetic side effects. Differences in the composition of the grafted cell preparation may contribute to these discrepancies. In particular, the number of serotonin neurons contained in the graft can vary greatly depending on the dissection of the fetal tissue. Importantly, serotonin neurons have the ability to store and release dopamine, formed from exogenously administered L-DOPA. Here, we have evaluated the effect of transplants containing serotonin neurons, or a mixture of dopamine and serotonin neurons, on L-DOPA-induced dyskinesias in 6-hydroxydopamine-lesioned animals. As expected, dopamine neuron-rich grafts induced functional recovery, accompanied by a 60% reduction in L-DOPA-induced dyskinesia that developed gradually over the first 10 weeks. Rats with serotonin-rich grafts with few dopamine neurons, in contrast, showed a progressive worsening of their L-DOPA-induced dyskinesias over time, and no functional improvement. The antidyskinetic effect of dopamine-rich grafts was independent of the number of serotonin neurons present. We conclude that serotonin neurons in the grafts are likely to have a detrimental effect on L-DOPA-induced dyskinesias in cases in which the grafts contain no or few dopamine neurons.

The 5-hydroxytryptamine transporter (5-HTT) regulates 5-hydroxytryptamine (5-HT) neurotransmission by removing 5-HT from the synaptic cleft. Emerging evidence from clinical and genetic studies implicates the 5-HTT in various neuropsychiatric conditions, including anxiety and depression. Here we report that a 5-HTT null mutant mouse line was generated by gene trapping that disrupted the sequence encoding the C-terminus of 5-HTT. This mutation resulted in significant reduction of 5-HTT mRNA and loss of 5-HTT protein. Brain levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid, were markedly decreased in C-terminus 5-HTT -/- mice, while 5-HT uptake or 5-HT content in platelets was absent. Behavioral phenotyping showed that C-terminus 5-HTT -/- mice were normal on a screen for gross behavioral, neurological, and sensory functions. In the tail suspension test for depression-related behavior, C-terminus 5-HTT -/- mice showed increased immobility relative to their +/+ controls. By comparison, a previously generated line of 5-HTT -/- mice lacking exon 2, encoding the N-terminus of the 5-HTT, showed abnormally high immobility in response to repeated, but not acute, exposure to the tail suspension test. In a novel, brightly-lit open field, both C-terminus 5-HTT -/- mice and N-terminus 5-HTT -/- mice displayed decreased center time and reduced locomotor activity compared with their +/+ controls. Both mutant lines buried significantly fewer marbles than their +/+ controls in the marble burying test. These findings further demonstrate the neurobiological functions of the 5-HTT and add to a growing literature linking genetic variation in 5-HTT function with emotional abnormalities.

alpha-Synuclein (alpha-Syn), a protein primarily localized in the presynaptic compartment of neurons, is known to regulate dopaminergic neurotransmission by negatively modulating dopamine transporter activity and regulating its trafficking to or away from the cell surface. Given the considerable homology between dopamine transporters and the serotonin (5-HT) transporter (SERT), we examined whether alpha-Syn could similarly regulate SERT function. Increasing expression levels of human alpha-Syn gradually decreased [(3)H]5-HT uptake by human SERT in cotransfected Ltk(-) cells, by diminishing its V(max) without changing its K(m), as compared to cells expressing only SERT. Biotinylation studies to label cell-surface proteins showed that alpha-Syn decreased the levels of SERT present at the plasma membrane. alpha-Syn and SERT were able to coimmunoprecipitate (co-IP), suggesting heteromeric complexes between these two proteins through direct protein-protein interactions. The negative modulation of SERT activity by alpha-Syn occurred through the non-Abeta-amyloid component (NAC) domain of alpha-Syn (aa58-107); DNA constructs encoding this region mimicked the full-length alpha-Syn protein by decreasing [(3)H]5-HT uptake by the transporter. Furthermore, only the constructs encoding the NAC domain of alpha-Syn prevented the co-IPs between full-length alpha-Syn and SERT, in both transfected cells and in rat solubilized lysates isolated from the prefrontal cortex. These studies suggest a novel physiological role for alpha-Syn in regulating SERT activity and may be of relevance in certain mental illnesses and in depression, in which SERT function is believed to be dysregulated.

S100B, a glia-derived calcium binding protein, exhibits strong neurite extension activity in cultured serotonergic neurons. Using S100B-knockout mice, we examined whether this protein possesses in vivo serotonergic trophic activity. The distribution of serotonergic fibers, determined by immunohistochemistry, in the brains of S100B-knockout mice was quite similar to that of wild-type mice. Furthermore, the content of serotonin and its metabolite 5-hydroxyindole-3-acetic acid in knockout mice was also indistinguishable from those of wild-type mice. Our findings argue against the hypothesis that S100B has a crucial role in neurite extension of serotonergic neurons.