The aim of this investigation was to determine whether i) affective touch has a role in mediating beneficial social influences on resilience to depression and ii) whether affective touch acts through specific skin CT afferents to enhance central serotonin function. To develop and validate the Touch Experiences and Attitudes Questionnaire (TEAQ), 117 items about experiences and attitudes to touch were completed online by 618 participants. Principal components analysis reduced this to 57 items and 6 factors. Three factors concerned touch experienced; in social situations (CST), in intimate relationships (CIT) and during childhood (ChT) and 3 factors concerned attitude to touch; in intimate relationships (AIT), with unfamiliar people (AUT) and in Skin Care (ASkC). The shortened TEAQ was completed by a second sample of 704 participants. Confirmatory factor analysis found the 6 factor structure to be a good fit of the data, suggesting the TEAQ to be valid and reliable. Participants completed some demographic questions and some questionnaires to determine their current psychiatric symptoms, social circumstances, recent life events, childhood adversity and personality alongside the TEAQ. Currently depressed participants had lower touch scores for all factors compared to healthy controls. Remitted depressed participants had significantly lower touch scores on all factors except CST, ASkC and AIT compared to healthy controls. A multiple regression analysis found neuroticism, satisfaction with social support, recent life events, CIT and childhood adversity (CHA) to be predictive of depression, whereas extraversion, number of social supports, ChT and CST, did not significantly predict depression score. Logistic regression analysis found ChT, CHA and neuroticism to predict vulnerability to depression, but not AIT or AUT. It was concluded that CIT was the most important aspect of affective touch for promoting resilience to depression. The CNS effects of pleasant and unpleasant touch were investigated using fMRI in healthy female volunteers. It has been hypothesised that a novel class of CT afferent fibres in hairy skin encodes affective touch. Therefore, CNS responses to pleasant stroking of the forearm with stroking of the fingers were compared. No differential CNS effects of forearm stroking over finger stroking were seen. Indeed, more brain regions were activated by pleasant brush stroking of the fingers which lack CT afferents. Pleasant brush responses in left inferior frontal gyrus were attenuated by tryptophan depletion. However, the midbrain raphe was activated by unpleasant brush stroking and de-activated by pleasant and this effect was abolished by tryptophan depletion. This study found little evidence that CT afferents in hairy skin have a specific role in affective touch and serotonin cells of the raphe appear engaged by unpleasant stimuli rather than pleasant. In conclusion, the results of the questionnaire study indicated touch (hugs, kisses, stroking) in intimate relationships may promote resilience to depression whereas touch with other social contacts does not, suggesting type of affective touch to be important. It is suggested that future studies of the role of current social support and of early adversity in depression should include assessments of the correlated dimension of affective touch. The fMRI study found little evidence for a specific peripheral touch receptor encoding pleasant affective touch. The median raphe nucleus was inhibited by pleasant touch and this is in keeping with the idea that that aversive stimuli activate serotonin projections to the forebrain but not that this is strengthened by affective touch. Further investigation is required to identify CNS mechanisms of affective touch.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

The discovery of stem and progenitor cells capable of ongoing neurogenesis in the adult mammalian brain has raised hope that we will one day be able to harness their intrinsic regenerative capacity following injury. Development of such therapeutic strategies relies on a comprehensive understanding of the underlying regulation of the neurogenic process. To this end, I show, in this thesis, that cultured post-natal hippocampal neural progenitor cells (NPCs) express a specific repertoire of connexins (Cx), a family of channel forming proteins critical for communication prior to the development of functional chemical synapses. I show that this pattern of Cx expression, specifically Cx43 and Cx45, is modulated by interaction with the extracellular matrix component laminin providing evidence of extracellular matrix-cell interaction in the regulation of intrinsic Cx expression and function in postnatal NPCs. In adult brain, I show, for the first time, that Cx45 localizes to all cell types of the neuronal lineage with the exception of the type 3 doublecortin (DCX)-positive NPCs. Using a loss of function approach, I show that this expression is required for the normal proliferation of type 1 nestin and glial fibrillary acidic protein-positive stem like NPCs but not for the differentiation or survival of their progeny in the adult hippocampus. With respect to exogenous pharmacological cues that influence hippocampal neurogenesis, this thesis also demonstrates that chronic treatment with a sub-set of selective serotonin reuptake inhibitor antidepressants, fluoxetine and escitalopram, increases the proliferation but not the survival of adult NPCs in healthy, non-depressed mice. Further, standard post-operative analgesia with the opiate buprenorphine inhibits the proliferation of DCX-positive adult NPCs and increases the survival of their progeny. Finally, over the course of the research for this thesis, it became clear that exposing research animals to even very subtle environmental changes can influence the basal neurogenic process. Ultimately this work further highlights the exquisite sensitivity of the regulation of what is already recognized to be a highly dynamic process and provides important insight into the neurogenic process that can be used to inform future therapeutic development and application.

Antidepressants are generally believed to exert their clinical efficacy by enhancing 5-HT transmission. Interestingly, sustained administration of selective serotonin (5-HT) reuptake inhibitors (SSRIs) strongly suppresses in the first few days the firing activity of 5-HT neurons in the dorsal raphe nucleus (DRN), thereby severely hampering the increase of 5-HT in target regions. Remarkably, the firing activity of 5-HT neurons gradually recovers over the time course of treatment and this recovery is believed to be accounted for by the desensitization of 5-HT1A somatodendritic autoreceptors. Here, we sought to investigate whether additional mechanisms might contribute to the dynamic regulation of excitability of 5-HT neurons during the course of SSRI treatments. Borrowing from the well-described homeostatic strengthening of glutamatergic synapses onto cortical pyramidal neurons following prolonged periods of inactivity, we hypothesized that a similar homeostatic-like regulation of synaptic strength might be operant on 5-HT cells during an SSRI treatment. To test this possibility, we used whole-cell electrophysiological recordings on acute midbrain slices to monitor glutamatergic synapses onto 5-HT neurons. We found that a two-day treatment with the SSRI citalopram induced a robust reduction in both the amplitude and frequency of AMPAR-mediated mEPSCs. We also show that this depression in synaptic strength, induced by an SSRI, is transient since excitatory drive onto 5-HT neurons was enhanced by 7 days of treatments. Altogether, these results document a dynamic regulation of glutamatergic synaptic transmission during the time course of a prolonged treatment with an SSRI. Further elucidation of the cellular and molecular mechanisms driving this synaptic plasticity might identify novel pharmacological target to shorten the delay of antidepressant action.

Production of the glucocorticoid hormone cortisol in response to a stressor is initiated by activation of the hypothalamic-pituitary-interrenal (HPI) axis in fish. Serotonin (5-HT) and negative feedback regulate cortisol production at the whole-animal level; the objective of the present thesis was to investigate their roles in regulating cortisol production by interrenal cells of rainbow trout (Oncorhynchus mykiss). Messenger ribonucleic acid (mRNA) for the 5-HT4 receptor was present in low abundance in interrenal cells. In addition, cortisol production was significantly increased for in vitro head kidney preparations incubated with 5-HT, and this elevated cortisol production was blocked by the 5-HT4 receptor antagonist 5-fluoro-2-methoxy-[1-[2-[(methylsulphonyl) amino] ethyl]-4-piperidinyl]-1h-indole-3-methylcarboxylate sulphamate (GR125487). Thus, 5-HT acts at the head kidney level to regulate cortisol production, probably via the 5-HT4 receptor. Chronic social stress did not appear to regulate the expression of key proteins involved in cortisol biosynthesis or corticosteroid receptors (CR). However, head kidney tissue incubated in vitro with cortisol for 2-8 h showed a reduction in adrenocorticotropic hormone-stimulated cortisol production compared to controls, suggesting the existence of an ultra-short-loop negative feedback mechanism. Thus, the high circulating levels of cortisol in trout experiencing chronic social stress may activate this ultra-short-loop negative feedback mechanism to suppress cortisol production at the head kidney level.

This study assessed the in vivo effects of various serotonin (5-HT) receptor modulators on 5-HT neurotransmission in the rat hippocampus. Vortioxetine, humanized-vortioxetine, and escitalopram blocked the 5-HT transporter, but similar to ipsapirone did not dampen the sensitivity of postsynaptic 5-HT1A receptors. Long-term administration of all treatments increased the tonic activation of postsynaptic 5-HT1A heteroreceptors, an effect common to all antidepressants. Vortioxetine decreased the function of the terminal 5-HT1B autoreceptor under high but not a low degree of activation, thus showing that its partial agonism led to increased 5-HT release and that long-term administration results in the desensitization of terminal 5-HT1B autoreceptors. Vortioxetine overcame the effects of 5-HT1B and 5-HT3 receptor agonists. This study was unable to determine the involvement of 5-HT7 receptor antagonism exerted by vortioxetine affects 5-HT neurotransmission. Therefore, vortioxetine would appear to exert different actions, via transporter and receptor activity, on the serotonergic system in the hippocampus, consistent with its unique pharmacological profile.

Multicellular organisms typically depend on O₂ for energy production to maintain normal cellular function, and even brief periods of O₂ deprivation may have fatal consequences. The aqueous environment is prone to changes in ambient water O₂ tension (PO₂) and thus the ability of fish to sense changes in water PO₂ and to elicit appropriate physiological responses is essential for their survival. Studies on fish O₂ chemoreception have identified neuroepithelial cells (NECs), which are characterized as having dense-cored vesicles containing serotonin (5-HT), as peripheral O₂ chemoreceptors. Upon exposure to hypoxia, isolated and cultured NECs in vitro depolarize, likely resulting in neurotransmitter release. However, to date there is no evidence that NECs are activated by hypoxia in vivo to initiate physiological responses such as the hypoxic ventilatory response (HVR), which is the focus of this thesis. Initial findings demonstrated that larval zebrafish fine-tune the HVR as early as 4 days post fertilization (dpf) and by 7 dpf, the HVR aids in O₂ uptake under hypoxic conditions. In addition, the HVR is multiphasic, with an initiation phase followed by a decline phase that gradually stabilizes above normoxic baseline values (Chapter 2). In the absence of tools to probe the hypoxia sensitivity of NECs in vivo, research focused on Merkel-like cells (MLCs), a newly proposed O₂ chemoreceptor in larval zebrafish. Using in vivo calcium imaging it was shown that MLCs are stimulated by hypoxia. Data suggest that MLCs are responsible for the initiation phase of the HVR, while peripheral sensory neurons (PSNs)/peripheral sensory ganglia (PSG) that innervate MLCs play a more important role in reducing ventilation during the decline phase of the HVR (Chapter 4). Attempts at identifying the putative neurotransmitter(s) involved in the O₂ signal transduction pathway revealed that adrenaline (AD), serotonin (5-HT), and dopamine (DA) are probable candidates (Chapter 4), though the presence of AD and DA within MLCs is yet to be confirmed. In addition, 5-HT likely plays a role in the central nervous system (CNS), integrating peripheral signals resulting in the final HVR (Chapter 3). Taken together, this thesis provides the first evidence of putative O₂ chemoreceptors responding to hypoxia in vivo and thus significantly advances models for O₂ signal transduction in larval zebrafish.

No description available.

Leaf and bark extracts of a Mexican medicinal plant, Byrsonima crassifolia (Malpighiaceae), exhibited spasmogenic effects on isolated rat fundus and biphasic effects on jejunum and ileum. Preliminary evaluations using rat fundus in Krebs solution indicated that the activity of a 2% acetic acid extract of eaves (HOAcE) could be split into two types: (i) high-efficacy, low-potency, n-butanol-extracted, pargyline- and 1-(1-naphtylpiperazine) (1-NP)-sensitive, atropine-insensitive activity, and (ii) low-efficacy, high-potency, ethyl acetate-extracted, pargyline-insensitive, atropine- and 1-NP-sensitive activity. HOAcE lacked muscarinic and nicotinic effects on rat jejunum and frog rectus abdominis. Serotonin (5-HT) and HOAcE curves in fundus were parallel and 5-HT potency was 6,037 times that of HOAcE (95% confidence limits: 4,624-7,852). The pD2 (affinity constant) for 5-HT was 7.96 (7.92-8.00) with pargyline added to the medium. 5-HT receptor-interaction kinetics using cholinergics and 5-HT agonists and antagonists was carried out. 1-NP competitively antagonized 5-HT. The 5-HT, antagonist s-(-)propranolol did not significantly antagonize 5-HT. The 5-HT2 blocker ketanserin noncompetitively antagonized 5-HT and α-Me-5-HT (pD'2 = 5.6 and 6.7, respectively). The 5-HT3 antagonist MDL-72222 inactivated only a small proportion of receptors (pD'2 = 6.46). Atropine did not significantly modify the curve of 5-HT while fluoxetine noncompetitively antagonized 5-HT (pD'2 = 5.8). 5-HT and α-Me-5-HT curves were biphasic indicating two receptor interactions (high and low affinity). High-affinity pD2 values for six different 5-HT agonists and 1-NP on rat fundus correlate well with reported rat brain (radioligand binding) pKd values at the 5-HT1C receptor (r = 0.94). Large scale extraction and fractionation of a methanol extract of leaves yielded two peaks of activity (Peak 1, lipophilic; Peak 2, polar). Peak 1 contained Compounds 1 to 7 (C1-C7); Peak 2 included C8-C15. Compound 1, C2, C3, C4, C10 and C11 were inactive while C8, C12 and C13 showed equivocal effects. Compound 5, C6, C7, C9, C14, C15, quercetin and gallic acid were active. Potencies were: C5 > C6 > C7 = quercetin > C9 > gallic acid. Efficacy (IA) was: C15 ≥ C14 > gallic acid > C9 > C5 > C7 > quercetin > C6. Compound 9 and its aglycone quercetin were partial agonists (C9 IA = 70%, pD2 = 6.35; quercetin IA = 60%, pD2 = 6.58). Compound 9 noncompetitively antagonized 5-HT (pD'2 = 6.10), while quercetin did not. Compound 14 and C15, the most active compounds, had similar response curves but these curves were not parallel to 5-HT. Spasmogenic ED50 values for C14 = 0.76 (0.38-1.54) μg/mL and C15 = 0.76 (0.41-1.42) μg/mL. Gram for gram 5-HT was 181 x C14 and 182 x C15.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Tereza Kalinová Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Mentor: Assoc. Prof. Jana Hroudová, Pharm.D., Ph.D. Title of diploma thesis: Effect of cholinesterase inhibitors on monoaminergic system and energy metabolism Cholinesterase (ChE) inhibitors play an essential role in the treatment of Alzheimer's disease (AD). They effect positively cognitive, functional and behavior symptoms of AD. Up to date, donepezil, rivastigmine and galantamine represent the only ChE inhibitors approved for AD treatment. The first ChE inhibitor was tacrine, which was withdrawn from market due to its toxicity and adverse effects. Recently, novel tacrine and 7-methoxytacrine (7-MEOTA) derivatives were synthetized and extensively investigated to find less toxic compounds affecting pathological mechanisms associated with development of AD. There is less known about effects of these drugs on mitochondrial functions and cellular energy metabolism. The aim of this project is to examine in vitro effects of ChE inhibitors on energy metabolism and cellular respiration, specifically on mitochondrial electron transport chain complexes and an enzyme of the citric acid cycle - citrate synthase. Inhibitory effects...