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

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

BMC neuroscience (2011-05-10)
Teruyuki Fukushima, Masayuki Tsuda, Takefumi Kofuji, Yuuichi Hori
ABSTRACT

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. 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. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IGEPAL® CA-630, for molecular biology
Sigma-Aldrich
Forskolin, from Coleus forskohlii, ≥98% (HPLC), powder
Sigma-Aldrich
IGEPAL® CA-630, viscous liquid
Sigma-Aldrich
Forskolin, For use in molecular biology applications
Supelco
IGEPAL® CA-630