Aims: (1) Analyze the roles of α7 and α4β2 nicotinic receptors (nAChRs) in nicotine sensitization in adolescent male and female rats neonatally treated with quinpirole as well as their effects on brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) 1 h and 24 h post drug treatment. (2) Analyze the effects of behavioral sensitization to nicotine on α7 and α4β2 nAChR density in the nucleus accumbens and dorsal striatum. Methods: Animals were neonatally treated with quinpirole (1 mg/kg) or saline from postnatal days (P)1-21. Beginning on P33, animals were ip injected with nicotine (0.5 mg/kg free base) or saline and tested every second day from P33-49. Approximately 30 min before injection, animals were ip injected with either the α7 nicotinic receptor (nAChR) antagonist methllycacontine (MLA; 2 or 4 mg/kg) or the α4β2 nAChR antagonist dihyro-β-erythrodine (DhβE; 1 or 3 mg/kg). Brain tissue was taken either 1 h or 24 h after the last day of testing. In a second experiment, animals were identically treated and brain tissue analyzed for nAChR density using the autoradiographic technique. Results: Neonatal quinpirole enhanced nicotine sensitization and the 3 mg/kg dose DhβE effectively blocked nicotine sensitization on Day 9 but enhanced the hypoactive response to nicotine on Day 1. MLA appears more important in the acute response to nicotine. Neonatal quinpirole sensitized the accumbal BDNF response to nicotine, but resulted in a decrease of accumbal mTOR. The nAChR density data will be presented. Conclusions: The α4β2 receptor played a critical role in the development of adolescent nicotine sensitization, and both nAChRs appear to be important in accumbal BDNF and in the mTOR response, demonstrating their important role in synaptic strength.

The cardiac ryanodine receptor (RyR2) plays a key role in excitation-contraction coupling (ECC). Mutations in RyR2 are known to be linked to the arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), a deadly disease which is characterized by a leak of calcium from sarcoplasmic reticulum and a decrease in calmodulin (CaM) binding. A novel drug, 84F2, shown to inhibit arrhythmias in RyR2-R176Q heterozygous CPVT mouse hearts (2.5 µg/kg), decrease spark frequency in cells derived from CPVT mice (IC50 = 35 nM), and inhibit RyR2 single channel activity at low nanomolar concentrations (IC50 = 8 nM). When CaM is added back to RyR2, 84F2's ability to inhibit channel activity is suppressed approximately 250 fold. A metabolite of 84F2, 78F3, is shown to also be active in the inhibition of RyR2. We propose that 84F2 decreases arrhythmias by binding to the CaM deficient RyR2, but does not affect normal ECC when CaM is present. This work characterizes for the first time a class of drugs whose inhibitory affects are dependent upon the removal of CaM from RyR2.

Semi-invariant natural killer T (iNKT) cells are critical components of the host immune response in peripheral tissues such as the lung, liver, and gut, and they play important roles in cancer, bacterial infections, autoimmunity, wound repair, and atherosclerosis. Tissue-resident iNKT cells exert their effects early in the developing immune response by rapidly producing a wide variety of cytokines and chemokines, and it was recently discovered that different tissues possess iNKT cell subsets that preferentially produce IFN-γ (NKT1), IL-4 (NKT2), or IL-17 (NKT17). Despite their critical role in the immune response, the mechanisms that regulate iNKT cell function in the periphery remain unclear. Signaling lymphocyte activation marker (SLAM) proteins are cell surface-expressed molecular switches that are expressed on all hematopoietic cells. The nine SLAM family receptors serve a variety of functions including promotion of cell-cell adhesion, regulation of cytokine production, co-stimulation, and inhibition. Importantly, SLAM family receptors are critical for the development of iNKT cells. Yet, numerous efforts to ascribe discrete roles of SLAM family receptors in iNKT cell function has proven difficult. We conducted a comprehensive analysis of SLAM family receptor co-expression on iNKT cell subsets in the lung, spleen, liver, and thymus and identified co-expression profiles that varied in a tissue and strain-dependent manner. Interestingly, we found that SLAM family receptor expression profiles varied among different iNKT cell subsets. In particular, we noted a close association of SLAMf6 expression with the NKT2 and NKT17 subsets in both the periphery and in the thymus. Further investigation using SLAMf6-deficient mice revealed a critical role for SLAMf6 in NKT2 and NKT17 subset development, and in iNKT IL-4 and IL-17 cytokine production in the periphery. This investigation also revealed that the SLAMf6high NKT2 and NKT17 subsets exhibited significantly higher proliferative capacity than the NKT1 subset and the NKT2 and NKT17 proliferation was dependent, in part, on SLAMf6 expression. Since Slam family genes are highly polymorphic, we next investigated whether these polymorphisms regulated iNKT function. We employed a B6.129 congenic mouse exhibiting impaired NKT cell function, in which a 6.6 Mbp 129/SvJ locus encompassing Slam genes was introgressed onto the C57BL/6 background. To test the hypothesis that Slam gene polymorphisms regulate iNKT cell function, we refined this genetic interval by generating B6.129 subcongenic lines and assessing iNKT cell function. Unexpectedly, we found that while Slam gene polymorphisms in this model do regulate iNKT cell function, the dominant regulator was in a 0.14 Mbp interval centromeric to the Slam genes. Further experimentation revealed that impaired iNKT cell development and function was associated with changes in the expression of Fcgr3 (Fc gamma receptor III) on iNKT cells, suggesting it as a novel candidate gene regulating iNKT cell function. Taken together, these data reveal for the first time a specific role for SLAMf6 on NKT2 and NKT17 subset development and function. In addition, these data identify Fcgr3 as a novel candidate gene that regulates iNKT cell subset development and cytokine production. Cumulatively, these data reveal the presence of discrete regulatory mechanisms at work in different iNKT subsets, a finding that has broad implications for our understanding of iNKT-cell mediated immunity.

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Errata pasted onto front end-paper. Includes bibliographical references (leaves 82-86). Describes the preparation and characterisation of a novel series of organoplatinum(II) complexes with hydrogen-bonding functionality.

"October 2001" Amendments (4 leaves) inserted inside back cover. Includes bibliographical references (leaves 215-266)