title: Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice.
abstract: BACKGROUND
  Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn.
  
  
  RESULTS
  To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC). Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP) under the control of the preproenkephalin (PPE) gene (penk1) promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side) compared with the contralateral (non-ligated side). These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors.
  
  
  CONCLUSIONS
  These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA), and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and modulation of nociceptive information.
authors.affiliation: Department of Physiology and Biological Information, Dokkyo Medical University, School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan. tfukushi@dokkyomed.ac.jp
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Fukushima
authors.forename: Masayuki
authors.initials: M
authors.lastname: Tsuda
authors.forename: Takefumi
authors.initials: T
authors.lastname: Kofuji
authors.forename: Yuuichi
authors.initials: Y
authors.lastname: Hori
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial; D004745:Enkephalins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D020671:Posterior Horn Cells / Q000378:metabolism / Q000502:physiology*; D011401:Promoter Regions, Genetic; D013116:Spinal Cord / Q000378:metabolism / Q000502:physiology*; D009435:Synaptic Transmission / Q000502:physiology
chemical_list: D004745:Enkephalins; D049452:Green Fluorescent Proteins