It is generally accepted that the neurotransmitter, serotonin mediates morphine-produced analgesia, however, it is not clear whether this mediation occurs at brain or spinal cord serotonin receptors. An issue that has not often been considered is the differential role that serotonin receptor types may play in morphine-produced analgesia. Paul and Phillips (1986) observed that pirenperone, a serotonin antagonist with a preferential affinity for the S2 receptor, attenuates morphine-produced analgesia. This result is particularly interesting because there are reportedly no S2 receptors in the spinal cord. The purposes of this dissertation were: to confirm the finding of Paul and Phillips, to localize the S2 receptors that mediate the anti-analgesic effect of pirenperone, and to test the hypothesis that pirenperone may exert its anti-analgesic effect through alpha-adrenergic receptors. In each of five experiments, tail-flick latencies (the time that it takes for each rat to withdraw its tail from a 52 C water bath) were measured 0, 30, 60, 90, and 120 min after drug injection. In Experiment 1, the analgesic effect of 10 mg/kg of morphine sulphate was challenged with 0.08, 0.16, and 0.24 mg/kg of pirenperone. Each dose of pirenperone attenuated morphine-produced analgesia. Moreover, each dose of pirenperone produced hyperalgesia in rats receiving no morphine. In Experiment 2, morphine-produced analgesia was challenged with 1, 3, and 10 mg/kg of ketanserin HCI. Only the very high 10 mg/kg dose ofketanserin significantly attenuated morphine-produced analgesia. Because ketanserin is pharmacologically similar to pirenperone but does not readily enter the central nervous system, this result indicates that central S2 receptors mediate the anti-analgesic effect of pirenperone and ketanserin. A third experiment demonstrated that 10 mg/kg of ketanserin did not block the analgesia produced by ketamine. Ketamine is thought to produce analgesia by a different mechanism than morphine. Thus, the attenuation of analgesia by S2 receptor blockers is not a general phenomenon, and it may be specific to morphine-produced analgesia and other analgesics that act on this system. Experiment 4 was designed to assess whether it is S2 receptors in the brain or in the spinal cord that mediate the anti-analgesic effect of S2 receptor blockade. The analgesic effect of morphine on tail-flick latencies was challenged with pirenperone in rats with spinal cords transected at the lower thoracic level and in sham-surgery comparison rats. Pirenperone attenuated morphine-produced analgesia in the sham-surgery group but not in the rats with transected spinal cords. These results indicate that brain S2 receptors mediate the attenuation of morphine-produced analgesia by pirenperone. In the fifth and final experiment, morphine-produced analgesia was challenged with 10 mg/kg of LY53857. LY53857 is an S2 antagonist which unlike pirenperone and ketanserin has no action at alpha-adrenergic receptors. Like pirenperone and ketanserin, LY53857 attenuated morphine-produced analgesia. This result supports the view that S2 receptorsmediate the anti-analgesic effects of pirenperone and ketanserin. Together, the results of these five experiments indicate that S2 receptors in the brain are important for opioid-mediated analgesia. This conclusion challenges the widely held view that only spinal cord serotonin receptors mediate morphine-produced analgesia. / Arts, Faculty of / Psychology, Department of / Graduate

No description available.

In 1985, Mendelson and Gorzalka proposed the dual role hypothesis of serotonergic modulation of lordosis behaviour. In this hypothesis it was proposed that serotonergic activity can either inhibit or facilitate lordosis behaviour. Specifically it was suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at 5-HT₁ receptors, whereas lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ receptors. The purpose of the following series of studies was both to confirm and to extend the dual role hypothesis. The intraperitoneal administration of the 5-HT2 antagonists pizotefin (1 mg/kg), cyproheptadine (1 mg/kg), metitepine (1 mg/kg), and ketanserin (1 mg/kg) were found to inhibit lordosis behavior in ovariectomized rats that had been primed with estradiol benzoate (EB) and progesterone (P). Pipamperone was ineffective. The 5-HT₂ agonist guipazine (3 mg/kg) was ineffective alone, but it reversed the inhibitory effects of pizotefin, cyproheptadine, and ketanserin. It did not reverse the effects of metitepine. The highly selective 5-HT₂ antagonist LY53857 (0.3 mg/kg) was also found to inhibit lordosis behaviour in female rats that had been primed with EB and P. The lordosis-inhibiting effect of LY53857 (1 mg/kg) in females primed with EB and P was reversed by quipazine (3 mg/kg). The nonselective 5-HT antagonist methysergide (7 mg/kg) was found to inhibit lordosis behavior 30 min after intraperitoneal administration to females treated chronically with EB, or with EB and P. However, methysergide was found to facilitate lordosis behavior 200 and 300 min after administration to female rats treated acutely with EB. In an analysis of dose response it was found that methysergide (0.02 - 7 mg/kg) administered 30 min prior to behavioural testing produced no facilitation of lordosis in females primed with EB. However, when administered 200 min prior to testing, methysergide (1 mg/kg) produced a significant facilitation of lordosis. The administration of the 5-HT₁ A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) inhibited lordosis behavior in ovariectomized rats primed with EB. 8-OH DPAT was ineffective at 0.01 mg/kg, whereas inhibition occurred at the 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg doses. In an evaluation of the effects of 8-OH DPAT on the expression of male sexual behaviour by females treated chronically with testosterone, 8-OH DPAT ( 1 mg/kg) increased the number of females mounting and significantly increased mount frequency. The 5-HT₁ A agonists ipsapirone (0.1 mg/kg) and gepirone (0.3 mg/kg) facilitated lordosis in females treated with EB. When administered at higher doses, ipsapirone (3.0 mg/kg) and buspirone (3.0 mg/kg) inhibited lordosis in rats treated with EB. In females treated with EB and P, ipsapirone (> 1.0 mg/kg), gepirone (> 0.3), and buspirone (> 0.3) inhibited lordosis behaviour. The newly developed 5-HT₁ A antagonist BMY 7378 (0.2 mg/kg) facilitated lordosis behaviour in females treated with EB. However, this facilitation was no longer apparent at the 5 mg/kg dose. BMY 7378 (0.04 - 5 mg/kg) was ineffective in females primed with EB and P. The 5-HTTB agonist 1 -(3-trifluoromethylphenyl)piperazine (TFMPP, 0.2 -5 mg/kg) was found to facilitate lordosis in females treated with EB. In females primed with EB and P, TFMPP (5 mg/kg) produced a significant inhibition of lordosis. The 5-HT₁ B agonist m-chlorophenylpiperazine (MCPP, 0.04 - 5 mg/kg) was ineffective in females primed either with EB or with EB and P. The 5-HT₃ Antagonist ICS 205-930 (5 mg/kg) was found to facilitate lordosis behaviour, whereas the 5-HT₃ Antagonist MDL 72222 (0.05 - 5 mg/kg ) was found to be ineffective in females primed with EB. The results of these studies tend to confirm that serotonergic activity can either inhibit or facilitate lordosis behaviour. It is suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at postsynaptic 5-HTTA and possibly 5-HT₃ Receptors. The lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ and possibly presynaptic 5-HT₁ B receptors. Finally, it is suggested that activity at somato-dendritic 5-HT₁ A autoreceptors may mediate facilitatory effects of low doses of 5-HT₁ A agonists. In closing, there is a discussion of the implications these results might hold for the understanding of the effects of serotonergic drugs on human behaviour. / Arts, Faculty of / Psychology, Department of / Graduate

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Chinese Medicine / Master / Master of Philosophy

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Determination of the physiologic roles of serotonin (5-HT) has long been hampered by the lack of compounds specific for certain of the 5-HT receptor subtypes. The objective of this dissertation was to characterize vascular serotonin receptors in certain arteries and to develop functional assays for the putative 5-HT₁(A) and 5-HT₂ receptors in vascular tissue to test novel compounds. Although 5-HT₁(A) receptor involvement in the 5-HT contraction of the canine basilar artery was previously reported, the 8-OH-DPAT (5-HT₁(A) specific agonist) EC₅₀ values in the canine, rabbit, guinea pig, and bovine basilar arteries studies were not consistent with the presence of 5-HT₁(A) receptors. Studies examining the 5-HT₂ selective antagonist ketanserin, several novel aryltryptamines with a range of affinities, and enantiomers of spiroxatrine, in the 5-HT-contracted rat aorta showed a good correlation between the aorta affinities and the affinities of these compounds at the [³H] ketanserin binding site (defined as 5-HT₂) in the rat frontal cortex. Comparison of the affinities of several known and novel compounds in the rat aorta and the rabbit femoral artery to the [³H] ketanserin site affinities in the frontal cortices of both species showed that the rabbit femoral artery 5-HT₂-like receptor was similar but not identical to either the rat aorta or the CNS sites from either species. The rabbit aorta and the rat femoral artery were then examined to determine if the 5-HT₂ receptor heterogeneity was species or vascular bed specific. The results from all four vascular tissues showed that no two tissues had identical responses to the compounds studied. The rat aorta appeared unique in the lack of agonist activity of RU24969 and the non-competitive antagonism of 5-HT by methysergide, but correlated to the CNS site for the affinities of all compounds. The major finding of the dissertation was the definitive evidence for vascular 5-HT₂ receptor heterogeneity; this subtype was previously thought to be homogeneous. Development of more selective compounds for 5-HT receptor subtypes may lead to greater understanding of the physiological roles of serotonin.

Motivation has been defined as a set of processes which enables organisms to overcome obstacles by energizing behavior in the pursuit of a goal. There are several important observations about motivated behavior which provide insight into the neural mechanisms underlying goal-directed motivation. First, motivation serves two important functions, as it both energizes behavior and also directs it toward or away from specific stimuli. Many of the behavioral tasks used to assay motivation in laboratory rodents do not specifically aim to measure these two distinct aspects of motivation. A second feature of goal-directed motivation is that it is sensitive to both costs and benefits of a given situation, enabling animals to make cost-benefit decisions. Again, many of the behavioral tasks which study cost-benefit decision making do not specifically aim to independently measure the impact of cost manipulations and benefit manipulations in an isolated manner. Here, I first develop behavioral measures which aim to specifically dissociate activational and directional effects of motivation. By characterizing a novel behavioral measure known as a Progressive Hold Down (Ph.D.) task, and using this task in parallel with a more traditionally used Progressive Ratio (PR) task, I show that methamphetamine robustly enhances activational effects of motivation, leading to increased response rates in both the Ph.D. and PR task, but mice are not more goal-directed in the Ph.D. task. I next develop and characterize two novel behavioral assays which are specifically used to examine effort and value contributions to cost-benefit decision making. The Concurrent Effort Choice (CEC) task measures how changes in effort levels impact decision making whereas the Concurrent Value Choice (CVC) task measure how changes in reward value impact decision making. Using these novel assays to examine specific processes important for goal-directed motivation, I carefully examine the role of manipulation of the Dopamine D2 receptor (D2R) in a mouse model which over-expresses the D2R within the striatum (D2R-OE), and the role of pharmacological manipulation of the Serotonin 2C receptor (5-HT2CR) with the functionally selective ligand SB242084. Whereas D2R-OE specifically impacts sensitivity to changes in effort levels which decrease overall levels of goal-directed motivation, selective modulation of the 5-HT2CR via treatment with SB242084 increases response vigor through enhanced dopamine release in the dorsomedial striatum, but this increase in response vigor does not alter sensitivity to effort or value changes when working for rewards. Together, these studies demonstrate the benefits of developing a more nuanced understanding of how specific manipulations impact motivated behavior by examining the specific underlying processes being altered.

The suprachiasmatic nucleus receives dense serotonergic projections from the raphe nuclei and this input has been implicated in the modulation of circadian rhythms. This input appears to have many functions including the transmission of non-photic information during the day and the modulation of photic information at night. However, it has emerged that this input may also be involved in the transmission of light information with activation of 5-HT2C receptors at night having a photo-mimetic effect. The studies described in this thesis aim to clarify the role of 5-HT2C receptors in the control of circadian rhythms in the rat model and compare their actions to light. The acute effects of 5-HT2C receptor agonist administration on clock gene expression were investigated in the rat SCN. Systemic administration of the 5-HT2A/2C agonist DOI to rats during early night induced c-fos, Per1 and Per2 expression in a manner similar to light. This response was time of day dependent with maximal induction occurring in the early night, and no response during the day. The role of 5-HT2C receptors in this response was confirmed with the use of the selective 5-HT2C receptor agonist RO-60 0175. The effect of 5-HT2C receptor activation on the phase of expression of various circadian rhythms including temperature, melatonin and clock gene expression in the SCN and periphery was examined. Both DOI administration and light exposure at night phase delayed rhythms of melatonin and temperature. Similarly, the selective 5-HT2C receptor agonist RO-60 0175 phase delayed rhythms of 6-sulphatoxymelatonin, a response which was antagonised by the 5-HT2C receptor antagonist SB-242084. The expression of functional and clock genes within the pineal was also phase delayed following both light and 5-HT2C receptor agonist administration. However, the phase of expression of clock genes within the SCN or liver did not shift in response to either a single nocturnal light pulse or agonist administration. To investigate the site of action of 5-HT2C receptor agonists, rat SCN explants were maintained in culture allowing exposure of agonists to denervated tissue. The acute effect of DOI administration at various circadian times on c-fos and Per1 expression was assessed. 5-HT2C receptor activation significantly increased Per1 expression when administered during early subjective night, but had no effect during either subjective day or late subjective night, similar to that observed in vivo. Finally, the suitability of immortalised rat SCN cells for investigation of the intracellular actions of 5-HT2C receptors in the circadian system was assessed. Using RT-PCR the expression of various serotonin receptors in the SCN2.2 cell line was compared with that observed in punches of adult rat SCN. The mRNA for 5-HT1B and 5-HT2A receptor was expressed in both the SCN2.2 cell line and the adult rat SCN. However, 5-HT2C receptor mRNA along with 5-HT3 receptor, 5-HT5A receptor and 5-HT7 receptor mRNA was expressed in the adult rat SCN tissue but not the SCN2.2 cells. These significant differences in serotonin receptor expression limit the usefulness of this cell line for further investigation. Together these experiments further implicate 5-HT2C receptors in the control of circadian rhythms. The role of these receptors appears limited to early night, with activation showing photo-mimetic responses. Furthermore, the location of action appears to be post-synaptic within the SCN, altering the core clock genes, which in turn phase delay various circadian rhythms. / Thesis(Ph.D.)-- School of Paediatrics and Reproductive Health, 2008

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences