Ms. Sandra Bruening. Ms. Bruening is currently examining the dynamics of cell proliferation in the developing hippocampus in receptor null mice. By using various rescue strategies, she is investigating the contribution of the abnormal proliferation to the anxiety phenotype of these mice. SSRIs such as fluoxetine are widely used in children and adolescents in the treatment of anxiety disorders and depression. However, the long term effects of SSRIs of such treatment are unknown. Ms. Bruening is studying the long-term and life-long effects of chronic administration of fluoxetine on the mouse CNS using molecular and behavioral techniques.
Ms. Jeanne Farrell. Ms. Farrell is identifying the cDNAs encoding these newly identified somatic Soluble Adenylyl Cyclase(sAC1 and sAC2 isoforms). She is currently characterizing the biochemical activities of sAC1 isoforms from brain and cloning sAC2 isoforms from brain and other somatic tissues by RT-PCR, RACE, and library screening.
Ms. Ann Gregus. Ms. Gregus is examining candidate gene expression in GluR5 subunit KO mice, a strain that does not develop morphine tolerance. Immunofluorescence studies of L4/L5 superficial spinal cord dorsal horn (SCDH) reveal that GluR5 KO mice exhibit phenotypic differences from their WT counterparts. The immunoreactivity of Calcitonin Gene-Related Peptide (CGRP), Protein Kinase C gamma (PKCg), and Dynorphin A in superficial SCDH is 2 fold greater in GluR5 KO than in WT mice. After morphine the amount of PKCgamma or Dynorphin A immunolabeling is unchanged in the GluR5 K) mice. These results suggest that the absence of the GluR5 receptor subunit results in a loss of the ability of morphine to regulate signaling of these proteins in SCDH neurons. How these changes are related to the failure of these mice to development of morphine tolerance is under investigation.
Ms. Amanda Weyerbacher. Ms. Weyerbacher is evaluating the role of NMDA receptors (NMDAR) in an inflammatory pain model (Complete Freund’s Adjuvant or CFA) using both pharmacological antagonism and a spatial spinal cord dorsal horn knockout we have developed. When CFA-induced mechanical allodynia is followed by repeated intrathecal (IT) injections of LY235959 (LY), a competitive NMDAR antagonist, the IT administration of LY was able to reverse allodynia at both 24 and 48 hours after CFA, but not at 96 hours post CFA administration. Her results indicate a time dependent loss of NMDAR antagonist efficacy, possibly resulting from the slow onset of an NMDAR-independent component of the CFA-induced mechanical allodynia. This model can be used in mice with a spatial deletion of the NMDAR in the spinal cord dorsal horn to directly test the hypothesis inflammatory pain involving both NMDA dependent and independent pain.
Mr. Daniel Han. Mr. Han is identifying the specific structural rearrangements of hallucinogen receptors that are triggered by ligand-dependent activation and can produce ligand-specific conformations of the regions that interact with the downstream proteins (PDZ domains) in the signaling cascade. He is focused on the rearrangements of a conserved motif, NPxxYx(5,6)F in transmembrane (TM) 7 that is responsible for the positioning of the C-terminus of the G protein coupled receptor in its interactions with PZD domains.
Sandra Garraway, PhD. Dr. Garraway is designing, testing and delivering siRNA’s targeted at genes known or suspected to be involved with injury-induced pain or opioid tolerance. She, together with Qinghao Xu a graduate student, has identified effective siRNA’s sequences that target the NR1 subunit of the NMDA receptor using an in vitro transfection system. The effective siRNA’s (and control sequences) have been incorporated into a recombinant adenoassociated viral vector and injected into the spinal cord dorsal horn of the mouse and the rat. These vector delivered sequences have successfully knocked down NMDA receptor expression in murine neurons as measured by in situ and NR1 protein expression. This research has provided new techniques for the examination of the role of NMDA receptors in pain and opioid tolerance.
Vilma Greene, PhD. Dr. Greene is determining whether our peptide antioxidants prevent mitochondrial calcium overload. To measure mitochondrial and cystoplasmic calcium, Vilma has successfully transfected Hela cells selectively expressing aequorin targeted to mitochondria or in the cytoplasm. Unfortunately, she has discovered that our antioxidant peptides inhibit coelenterazine, the cofactor necessary for aequorin to interact with calcium and produce a luminescent signal. She is now trying to use other methods, including the fluorescent dye rhodamine-2, to measure mitochondrial calcium. She is also exploring other fluorescent approaches such as the various “chameleons” that can be targeted to different organelles.
Felix Kim, PhD. Dr. Kim has demonstrated that sigma1 receptors do not influence opioid receptor binding, but can significantly influence the ability of opioids to activate receptors as determined by a GTPgammaS binding assay. This effect is shown by shifts of the dose-response curves by agonists and antagonists. He has extended these studies to explore putative binding domains on the sigma1 receptor that interact with opioid receptors and has demonstrated that the two proteins co-immunopurify.
Diane Lane, PhD. is conducting electron microscopic immunolabeling studies to determine whether chronic intermittent administration of escalating doses of morphine produced changes in the subcellular distribution of the Glu R1 subunit of the AMPA receptor in the VTA. The morphine treated group showed an overall increase in total dendritic Glu R1 labeling mainly in dopaminergic dendrites as compared to controls. Despite this increase, there was a decrease in the dendritic plasma lemmal Glu R1 distribution in morphine treated rats. These results provide ultrastructural evidence that chronic intermittent morphine evokes changes in both the expression and surface trafficking of AMPA receptor subunits in dopaminergic neurons of the VTA.