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Open Access Research article

Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

Teruyuki Fukushima1*, Masayuki Tsuda1, Takefumi Kofuji2 and Yuuichi Hori1

Author Affiliations

1 Department of Physiology and Biological Information, Dokkyo Medical University, School of Medicine, Kitakobayashi 880, Mibu, Tochigi 321-0293, Japan

2 Radioisotope Laboratory, Kyorin University, School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan

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BMC Neuroscience 2011, 12:36  doi:10.1186/1471-2202-12-36

Published: 7 May 2011

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.