Selective Serotonin Reuptake Inhibitors (SSRIs) are the first-line treatment for major depression, but require several weeks to elicit a clinical effect. One mechanism that may underlie this delay in SSRI action implicates the gradual desensitization of 5-HT1A autoreceptors, leading to enhanced firing and increasing serotonin (5-HT) at the synapse. I hypothesized that in absence of 5-HT1A autoreceptors, fluoxetine (FLX) would improve behavior faster and more effectively. To specifically knock out 5-HT1A receptors in 5-HT neurons, we crossed TPH2-CREERT2 and flx5HT1A-YFP mice to generate the flx1A mice, a tamoxifen-inducible conditional knockout. Tamoxifen-induced recombination in adult flx1A-/- mice induced YFP expression and reduced 5-HT1A receptor levels by over 90%, specifically in TPH-positive cells of the raphe. To test the response to sub-chronic SSRI treatment, the mice were treated for 9 days with SSRIs FLX or escitalopram and examined for anxiety and depression-like behavior using multiple tests, including the novelty suppressed feeding test (NSF) that responds to chronic but not acute SSRI treatment. Subchronic FLX treatment had no effect in flx1A +/+ control mice, but resulted in an unexpected anxiogenic effect in flx1A -/- littermates, in both the NSF and elevated plus maze tests. Subchronic treatment with escitalopram also increased anxiety-like behavior in the NSF in flx1A-/-, but not wild-type mice. To determine whether FLX treatment differentially affected brain activity in these mice, the number of FosB-stained cells was determined as an index of chronic activation. In flx1A -/- vs. +/+ mice, the number of FosB-positive cells was reduced in several brain regions linked to anxiety and depression including hippocampus and entorhinal cortex, and FLX III treatment reduced activation in the flx1A +/+ compared to -/- mice these areas and in 5-HT neurons of the median raphe nucleus. These results suggest that in the absence of 5-HT1A autoreceptors, SSRIs have a pro-anxiety effect that may involve reduced inactivation anxiety-related brain regions.

The synthesis of the pineal hormone, melatonin, is finely regulated by the pineal enzyme serotonin N-acetyltransferase (NAT). In the absence of light, the activity of NAT is markedly enhanced by the release of nor-adrenaline from sympathetic nerve endings in the pineal. Exposure of animals to light during darkness causes a sudden and dramatic reduction in the activity of NAT. The present study investigated a possible mechanism for this sudden decline in NAT activity. These investigations included the determination of the effects of S-adenosylmethionine (SAM), adenosine nucleotides and calcium on NAT activity. In vitro experiments using SAM showed that pineals pre-incubated with SAM prior to adrenergic stimulation did not significantly alter NAT activity or pineal indoleamine metabolism. However, measurement of pineal cyclic AMP showed that SAM exposure reduced the adrenergic-induced rise in pineal cyclic AMP. Experiments using adenosine 5'-monophosphate (5'-AMP) showed that this nucleotide enhanced both dark- and isoproterenol-induced NAT activity. Adenosine 5'-triphosphate (A TP), on the other hand, reduced NAT activity with a concomitant reduction in pineal indoleamine metabolism. Exposure of isoproterenol-stimulated pineals in organ culture to propranolol resulted in a marked rise in ATP and adenosine 5'-diphosphate (ADP) synthesis accompanied by a decline in 5'-AMP levels as compared with pineals treated with isoproterenol alone. This then implies that exposure of animals to light could cause a change in pineal nucleotide levels. Since nucleotide levels are also controlled by calcium, experiments were carried out to determine the effect of calcium on pineal NAT activity. These experiments showed that ethyleneglycol-bis-N,N,N,N,-tetraacetic acid (EGTA) enhanced NAT activity whilst calcium reduced the activity in pineal homogenates, implying that calcium may act directly on NAT to regulate its activity. Exposure of pineal glands in organ culture to the calmodulin antagonist R24571 caused a rise in pineal cyclic AMP levels with a concomitant decrease in cAMP-phosphodiesterase activity. This was, however, accompanied by a decline in Nacetyl serotonin and melatonin synthesis. These findings implicate a number of factors in the regulation of pineal NAT activity. A mechanism for the regulation of pineal NAT is proposed.

The effect of tricyclic antidepressants (TADs) on a variety of pineal functions was assessed. TADs affected the uptake of ³H-5HT into bovine pineal slices within a particular concentration range of these drugs, DESI, CLOMI and IMI appeared to inhibit uptake slightly, within a limited concentration range. Surprisingly, DESI appeared to be a relatively potent 5HT uptake inhibitor. The 5-HT re-uptake system in the pineal probably differes from that in brain tissue. TADs had a marked effect on the metabolism of ³H-5HT in the rat pineal, in an organ culture system, MEL and N-acetylserotonin synthesis increased for the first 11 days and thereafter a slight decrease was observed. HTOH and HIAA also showed an initial increase followed by a slight decrease in synthesis. The synthesis of MTOH and MIAA was decreased. The possibility that TADs could affect HIOMT and SNAT synthesis and thereby change the metabolic pattern of 5-HT was investigated. TADs appeared to stimulate SNAT initially and thereafter a slight decrease from peak activity was observed. This is probably due to stimulation followed by development of subsensitivity of β-receptors, HIOMT activity also appeared to be affected by TADs. The existence of two types of HIOMT is suggested. There is a possibility that these changes in the metabolism of 5-HT could be implicated in the mechanism of action of TADs. / KMBT_363 / Adobe Acrobat 9.53 Paper Capture Plug-in

Despite substantial progress in the area of depression research, the current treatments for Major Depressive Disorder (MDD) remain suboptimal. Therefore, various medications are often used as augmenting agents in pharmacotherapy of treatment-resistant MDD. Despite the relative clinical success, little is known about the precise mechanisms of their antidepressant action. The present work was focused on describing the effects of three drugs with distinctive pharmacological properties (pramipexole, aripiprazole, and quetiapine) on function of the monoaminergic systems involved in the pathophysiology and treatment of MDD. Reciprocal interactions between the monoamines serotonin, norepinephrine, and dopamine systems allow the drugs targeting one neuronal entity to modify the function of the other two chemospecific entities. Electrophysiological experiments were carried out in anaesthetized rats after 2 and 14 days of drug administration to determine their immediate and the clinically-relevant long-term effects upon monoaminergic systems. Pramipexole is a selective D2-like agonist with no affinity for any other types of receptors. It is currently approved for use in Parkinson’s disorder and the restless leg syndrome. Long-term pramipexole administration resulted in a net increase in function of both dopamine and serotonin systems. Aripiprazole is a unique antipsychotic medication. Unlike all other representatives of this pharmacological class that antagonize D2 receptor, this drug acts as a partial agonist at this site. Chronic administration of aripiprazole elevated the discharge rate of the serotonin neurons, presumably increasing the overall serotonergic neurotransmission. Like aripiprazole, quetiapine is one of three atypical antypsicotic drugs approved for use in MDD. Prolonged administration of quetiapine led to a significant increase in both noradrenergic and serotonergic neurotransmission. Importantly, the clinically counter-productive decrease in the spontaneous firing of catecholaminergic neurons, induced by SSRIs, was overturned by the concomitant administration of both aripiprazole and quetiapine. The increase in serotonergic neurotransmission was a consistent finding between all three drugs studied herein. In every case this enhancement was attained in a distinctive manner. Understanding of the precise mechanisms leading to the amplification/normalization of function of monoamines enables potential construction of optimal treatment strategies thereby allowing clinicians greater pharmacological flexibility in the management of depressive symptoms.

Cariprazine is a novel dopamine (DA) and serotonin (5-HT) partial agonist with an in vitro receptor affinity profile that endows it with the potential to be used successfully in the treatment of both unipolar and bipolar disorders. The objective of this study was to determine whether in vitro findings with cariprazine lead to functional alterations of monoamine systems in the intact rat brain. In vivo electrophysiological recordings were carried out in male Sprague-Dawley rats under chloral hydrate anesthesia. Dorsal raphé nucleus (DRN), locus coeruleus (LC), and hippocampus cornu ammonis region 3 (CA3) pyramidal neurons were recorded and cariprazine was administered systemically by intravenous injection or locally through iontophoresis. In the DRN, cariprazine induced a complete inhibition of the firing of 5-HT neurons, which was fully reversed by the selective 5-HT1A antagonist WAY100.635. In the LC, the inhibitory effect of the preferential 5-HT2A agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) was reversed by cariprazine with an ED50 value of 67 µg/kg, i.v., and it did not block the inhibitory effect of the α2-adrenergic agonist clonidine. In the hippocampus, when cariprazine was administered by iontophoresis, it inhibited the firing of pyramidal neurons, but it did not dampen the suppressant effect of 5-HT. These results indicate that, in vivo, cariprazine acts as a 5-HT1A agonist in the DRN, as an antagonist on 5-HT2A receptors controlling the firing of NE neurons, and is a full agonist at 5-HT1A receptors located on pyramidal neurons of the hippocampus. The modulatory actions of cariprazine on the 5-HT and NE systems may contribute to its reported effectiveness in depressive episodes.

Indiana University-Purdue University Indianapolis (IUPUI) / The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Behavioural sensitization models are useful for understanding many disorders, including obsessive-compulsive disorder and drug addiction. Many of these models are produced by sensitization of dopamine neurotransmission, resulting in behaviours which include increased locomotor activity. Alterations to dopamine-mediated locomotor sensitization may be possible via activation of serotonergic neurotransmission, and there is evidence to suggest this may be through repeated activation of serotonin 1A receptors. The current study examines the development of locomotor sensitization in an animal model via repeated exposure of both a dopamine (D2R/D3R) and serotonin 1A (5-HT1A) agonist. To examine this, male Long-Evans rats were exposed to 10 injections of a combination of different doses of quinpirole and 8-OH-DPAT and tested in activity chambers for locomotor stimulation (measured by total distance travelled). Animals were then exposed to challenges of quinpirole, and 8-OH-DPAT and tested again for locomotor activity. Results showed that high doses of quinpirole or 8-OH-DPAT induced locomotor sensitization. However, when the two drugs were co-administered, 8-OH-DPAT displayed some initial disruption of quinpirole-induced sensitization. Animals sensitized to either quinpirole or 8-OH-DPAT did show higher locomotion when challenged with the drug to which they were sensitized. However, simultaneous quinpirole and 8-OH-DPAT sensitization seemed to prevent maximal responding when challenged with quinpirole. In all, our data suggests that sensitization to quinpirole and 8-OH-DPAT is occurring via separate neural mechanisms, with 5-HT1A agonism interfering with development of dopaminergic (D2R) sensitization. / Thesis / Master of Science (MSc)

Background: There is a potential drug interaction between linezolid and antidepressants resulting in serotonin syndrome. Thus, clinicians often avoid this drug combination. However, little empirical data exists to support this avoidance. The objective of this study was to describe the risk of serotonin syndrome in patients receiving linezolid and how this risk changed with concomitant antidepressant use. Methods: A population based retrospective cohort study was conducted using the administrative databases at ICES. The patient population consisted of outpatients aged 66 years or older who were prescribed oral linezolid of any duration from 2014 to 2021 in Ontario, Canada. Patients who were also taking antidepressants during linezolid treatment were compared to patients not on antidepressants during linezolid treatment. The primary outcome was clinically significant serotonin syndrome requiring emergency room visit or hospitalization based on physician diagnosis, Sternbach criteria or Hunter criteria within 30 days of starting linezolid. Secondary outcomes included altered mental status, hospitalization and death due to any cause within 30 days. Results: Of 1,134 patients who were prescribed linezolid, 215 (19.0%) patients were also taking antidepressants. Less than 6 (<0.5%) patients had serotonin syndrome. The proportion of patients with serotonin syndrome was numerically lower in the antidepressant group. In a propensity score matched cohort, the adjusted risk difference for serotonin syndrome in the antidepressant group minus the no antidepressant group was -1.2% (95% CI -2.9% to 0.5%). The risk of altered mental status, hospitalization and death were similar between the two groups. Conclusions: The risk of serotonin syndrome was low in patients taking linezolid. Concurrent antidepressants did not significantly increase the risk of serotonin syndrome. These findings suggest that linezolid can be safely used in patients also on antidepressants when indicated. / Thesis / Master of Science (MSc) / Linezolid is an antibiotic that can potentially cause serotonin syndrome as an adverse effect when combined with antidepressants. In serotonin syndrome, dysfunction of the nervous system leads to a variety of symptoms that can be life threatening. This study examined people in Ontario aged 66 years or older who were prescribed linezolid from 2014 to 2021 to describe the risk of serotonin syndrome due to linezolid and how antidepressants change this risk. Patients were followed for 30 days from start of linezolid treatment to determine if they had serotonin syndrome based on diagnoses in emergency room or hospital visit records. Of 1,134 patients in the study, 215 (19.0%) patients took antidepressants. The risk of serotonin syndrome was low at less than 0.5%. This risk was not significantly different in patients on antidepressants when compared to those who were not. Therefore, linezolid is likely safe for patients receiving antidepressants.

Die unipolare Depression ist eine der häufigsten psychiatrischen Erkrankungen und geht mit einem hohen Leidensdruck für die Betroffenen einher. Die Symptomatik der Depression besteht v.a. aus gedrückter Stimmung, Interessenverlust und Antriebslosigkeit und führt bei den Betroffenen zu Einbußen in der sozialen und beruflichen Funktionalität. Daneben leiden die Patienten aber auch unter wechselnden Therapieversuchen u.a. aufgrund von fehlendem Ansprechen auf Medikamente. Trotz intensiver Forschung sind die Mechanismen der Krankheitsentstehung und die Wirkweise der antidepressiven Therapie nur teilweise verstanden. Genetische Studien identifizierten einige Suszeptibilitätsgene, die jedoch die Erblichkeit der depressiven Erkrankung nicht ausreichend erklären. Diese „missing heritability“ könnte durch epigenetische Faktoren wie z.B. Veränderungen in der DNA-Methylierung bedingt sein. Neben einer ätiopathogenetischen Rolle kommen epigenetische Modifikationen auch als Marker zur Prädiktion des Therapieerfolgs sowie als Korrelat des biologischen Wirkmechanismus der antidepressiven Therapie infrage. Die vorliegende Arbeit untersuchte daher die Pharmakotherapie-Epigenetik eines Suszeptibilitätsgens (SLC6A4, 5 HTT), das den Serotonin-Transporter kodiert. Hierbei wurde die wechselseitige Beziehung zwischen der antidepressiven Pharmakotherapie und der DNA-Methylierung von neun CpG-Dinukleotiden des Serotonin-Transporter-Gens in Hinblick auf den Therapieerfolg analysiert. Dabei kamen molekularbiologische Methoden wie die Bisulfitsequenzierung zur Ermittlung der DNA-Methylierung sowie psychometrische Diagnostik zur Quantifizierung des Therapieansprechens zum Einsatz. Stationär aufgenommene Patienten mit einer aktuellen depressiven Episode wiesen einen eher geringen durchschnittlichen Methylierungsgrad des Serotonin-Transporter-Gens von 5,5 % auf, wobei die Werte der einzelnen CpG-Dinukleotide von 1,6 % bis 9,8 % reichten. Die mittlere Methylierung zu Studienbeginn sowie die Methylierung der einzelnen CpG-Dinukleotide zeigte dabei keine Korrelation mit dem Therapieerfolg, d.h. der Änderung im Hamilton-Score. Patienten mit hoher und niedriger Methylierung unterschieden sich nicht eindeutig im Wochenverlauf der Hamilton-Scores und auch eine Einteilung der Patienten nach Response bzw. Remission ergab keine Unterschiede der SLC6A4-Methylierung in den jeweiligen Gruppen. Der Methylierungsstatus des 5 HTT-Gens sowie die Methylierungswerte einzelner CpG-Dinukleotide sind demnach diesen Daten zufolge nicht zur Prädiktion des Therapieerfolgs geeignet. Nach sechswöchiger Psychopharmakotherapie lag die mittlere Methylierung bei 6,0 %, wobei keine signifikante Veränderung nachgewiesen werden konnte. Einzelne CpG-Dinukleotide zeigten jedoch einen Trend zu einer Methylierungszunahme. Die mittlere Methylierungänderung korrelierte nicht mit der Änderung des Hamilton-Scores, nur für CpG6 und CpG9 ergaben sich nominell signifikante positive Korrelationen. Gruppiert nach Response bzw. Remission konnte kein signifikanter Unterschied der mittleren Methylierungsänderungen nachgewiesen werden. Bei Therapie-Respondern schien die Methylierung an den meisten CpG-Dinukleotiden zuzunehmen. Lediglich bei CpG6, CpG8 und CpG9 wiesen Non-Responder eine stärkere Methylierungszunahme auf. Auffällig war v.a. CpG1, das bei Non-Respondern eine nominell signifikante Methylierungsabnahme zeigte. Demnach besteht möglicherweise ein Zusammenhang zwischen der Methylierungsänderung einzelner CpG-Dinukleotide des 5 HTT-Gens unter antidepressiver Therapie und dem Therapieerfolg der Patienten. In Bezug auf die Pharmakotherapie hatten ausschließlich SSRI einen signifikanten Einfluss auf die Änderung der SLC6A4-Methylierung. Dabei zeigten Patienten unter SSRI-Therapie eine deutliche Methylierungszunahme, die synergistisch mit der Blockade des Serotonin-Transporters wirken könnte. Epigenetische Modifikationen des 5 HTT-Gens kommen folglich als molekularer Wirkmechanismus dieser Behandlung in Betracht und implizieren neue Ansätze für innovative Pharmakotherapeutika. Die vorliegende Arbeit liefert somit einen Beitrag zum Verständnis der zugrundeliegenden molekularbiologischen Prozesse der antidepressiven Therapie. Zur Sicherung und Replikation der gefundenen Ergebnisse sind jedoch weitere Studien mit größeren und genauestens charakterisierten Stichproben nötig. / Unipolar Depression is one of the most frequent psychiatric diseases and is characterized by an enormous strain for the persons affected. The symptoms of depression are composed of depressed mood, loss of interest and reduced energy and cause deficits in social and professional functionality. Additionally, patients suffer from changing treatment attempts due to non-response to medication. Despite intensive research, mechanisms of disease development and antidepressant action are only partly understood. Genetic studies identified several susceptibility genes which however cannot completely explain the heritability of depressive disorder. This „missing heritability” could be due to epigenetic factors like e.g. changes in DNA methylation. Besides an etiopathogenetic role, epigenetic modifications can also be considered as predictive markers of therapy success as well as biological mechanisms of antidepressant therapy. Thus the present study investigated the pharmacotherapy-epigenetics of a susceptibility gene (SLC6A4, 5 HTT) which codes for the serotonin transporter. The reciprocal relation between antidepressant pharmacotherapy and DNA methylation of nine CpG dinucleotides of the serotonin transporter gene was analysed with regard to therapy success. Therefore molecular biological methods like bisulfite sequencing to determine DNA methylation as well as psychometric diagnostics to quantify therapy response were used. In-patients with acute depressive episode showed a rather low mean methylation level of the serotonin transporter gene of 5,5 % while values of the individual CpG dinucleotides ranged from 1,6 % to 9,8 %. The mean methylation at baseline as well as the methylation of the individual CpG dinucleotides did not show a correlation with therapy success that is the change in Hamilton score. Patients with high and low methylation did not differ in weekly Hamilton scores and a classification of patients by response or remission status did not yield any difference in SLC6A4 methylation between the respective groups. According to this data, the methylation status of the 5-HTT gene as well as the methylation values of the individual CpG dinucleotides are therefore not applicable for the prediction of therapy success. After six weeks of psychopharmacotherapy the mean methylation was 6,0 % whereas no significant change could be detected. However, individual CpG dinucleotides showed a trend towards an increase of methylation. The mean change in methylation did not correlate with the change in Hamilton score, only for CpG6 and CpG9 nominally significant positive correlations were demonstrated. Grouped by response and remission respectively, no significant difference in mean methylation change was detected. The methylation of the most CpG dinucleotides seemed to increase in therapy responders. Only at CpG6, CpG8 and CpG9 non-responder revealed a stronger increase in methylation. Noticeable above all was CpG1, that showed a nominally significant decrease in methylation in non-responders. Therefore a relation possibly might exist between methylation change of individual CpG dinucleotides of the 5-HTT gene under antidepressant therapy and therapy success of the patients. With regard to pharmacotherapy only SSRI had a significant influence of the change in SLC6A4 methylation. Patients under SSRI therapy showed a clear increase in methylation, which could act synergistic with the blockade of the serotonin transporter. Therefore epigenetic modifications of the 5-HTT gene should be considered as molecular mechanism of action of this treatment and implicate new approaches for innovative pharmacotherapeutics. The present work thus provides a contribution to the understanding of underlying molecular biological processes of antidepressant therapy. To assure and replicate the detected results further studies with larger and precisely characterized samples are necessary.