GENETIC STUDIES OF OPIOID RECEPTOR FUNCTION

Project Details

Description

The long-term goals of this project are to use genetic approaches to
investigate the functions of opioid receptors. Three distinct opioid
receptor cDNAs have recently been cloned that represent the major receptor
classes previously defined pharmacologically. The availability of these
probes thus provides a unique opportunity to apply molecular approaches,
in combination with biochemical, behavioral and cellular methods, to
examine the roles of endogenous opioids in development, pain perception
and neuroimmune function. Experiments addressing three specific aims are
proposed. In the first, we will determine the ontogeny of the delta, mu,
and kappa opioid receptors using in situ hybridization and
immunocytochemistry. The distribution and timing of receptor transcript
and protein expression will be investigated and compared to existing
binding data and to the expression patterns of endogenous ligands
including POMC, enkephalin, and dynorphin. These studies are designed to
identify putative sites of endogenous opioid action during development, to
determine the specific receptor classes active at those sites, and provide
a basis for examining potential developmental defects caused by receptor
inactivation. In the second aim, we will use gene targeting procedures to
inactivate the delta, mu, and kappa opioid receptor genes. We will isolate
mouse opioid receptor genomic clones and use the clones to construct
targeting vectors containing selectable markers. We will apply methods,
already successfully used in our laboratory, to substitute one opioid
receptor null allele for one wild-type allele in embryonic stem cells.
These cells will be injected into blastocysts and germ-line chimeras will
be identified, which will allow us to generate new animal strains lacking
opioid receptors. These strains will be examined for developmental
abnormalities at the sites of normal receptor expression identified in Aim
1 and mated to produce strains with multiple mutant receptors. In
addition, we will use radioligand binding assays and assays that
differentiate in vivo each receptor subtype to determine whether, in an
individual knock-out, all subtypes of each receptor class are coordinately
lost. We will also compare the patterns of expression of non-mutated
opioid receptor genes in specific receptor mutants with the normal
patterns to determine whether compensation at the transcriptional level
accompanies any of the three mutations. In the third aim, we will examine
the behavioral and physiological consequences of receptor inactivation
using two well-established experimental models. First, a series of
behavioral assays of opioid function will be used to monitor the effect of
receptor inactivation on pain perception. These studies will focus on
stress-induced analgesia and the analgesic actions of opiate drugs such as
morphine. In the second, we will investigate a newly discovered role of
endogenous opioids in regulating macrophage chemotaxis after injury to the
developing brain. We will analyze the migratory behavior of brain
phagocytes from normal and mutant mice following visual cortex ablation
using markers for macrophages and microglia and in vitro assays of
chemotaxis. Taken together, these studies should establish clear roles for
each opioid receptor class in specific biological processes associated
with behavior and drug addiction.
StatusFinished
Effective start/end date9/1/946/30/02

Funding

  • National Institute on Drug Abuse
  • National Institute on Drug Abuse
  • National Institute on Drug Abuse
  • National Institute on Drug Abuse
  • National Institute on Drug Abuse
  • National Institute on Drug Abuse: $412,942.00
  • National Institute on Drug Abuse
  • National Institute on Drug Abuse: $400,916.00
  • National Institute on Drug Abuse

ASJC

  • Genetics
  • Molecular Biology

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