Short communicationNerve growth factor regulates synaptophysin expression in developing trigeminal ganglion neurons in vitro☆
Introduction
Primary sensory neurons with cell bodies in the trigeminal ganglion (TG) carry both nociceptive and non-nociceptive information from the craniofacial region, including several clinically significant structures, such as the meninges, temporomandibular joint, and teeth. The TG system remains plastic after birth, with as many as 90% of neurons innervating a specific target destined to be eliminated during the first 90 days of postnatal maturation (O’Connor and Van der Kooy, 1986). However, the cellular mechanisms of postnatal maturation of TG neurons remain largely unknown.
Only a few previous studies addressed the role of neurotrophic factors in postnatal maturation of TG neurons. These studies indicated that one member of the neurotrophin family of growth factors, nerve growth factor (NGF), is a major player in the establishment of trigeminal target innervation (Naftel et al., 1994, Qian and Naftel, 1996, Nosrat et al., 1997, Nosrat et al., 2001, Yang et al., 2006). NGF is expressed by peripheral targets of TG neurons (Nosrat et al., 1997, Nosrat et al., 2001), and increases the cell body diameter and neurite outgrowth of newborn TG neurons in vitro (Lillesaar et al., 2003). Deprivation of NGF during early postnatal development results in a decrease in the number of sensory neurons innervating the tooth pulp in adult rats, with small-diameter cells and unmyelinated axons being most severely affected (Qian and Naftel, 1996).
Previous studies have demonstrated that synaptophysin (synaptophysin I; Syp), an integral membrane protein of synaptic vesicles, is involved in several crucial aspects of synaptic vesicle trafficking, including the initiation of neurotransmitter release (Valtorta et al., 2004). Moreover, Syp plays an important regulatory role in activity-dependent competitive synapse formation in cultures of hippocampal neurons (Tarsa and Goda, 2002). The goal of the present study was to examine whether NGF could regulate synaptophysin in developing TG neurons, and thereby promote activity-dependent maturation of synaptic connections in trigeminal pathways.
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Animals
Postnatal day (P) one Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were used for this study. All procedures were approved by the Institutional Animal Care and Use Committee of the Oregon Health and Science University, and conformed to the Policies on the Use of Animals and Humans in Neuroscience Research approved by the Society for Neuroscience.
Preparation of TG cultures
Rat pups were deeply anesthetized by intraperitoneal injection of Euthasol (0.1 mg/kg) and decapitated. TGs were dissected and
Results
We have examined the effects of nerve growth factor (NGF) on neuronal expression of synaptophysin (Syp), as an index of synapse formation during early postnatal maturation of TG neurons. P1 TG cultures were treated with either PBS vehicle or 100 ng/ml NGF as previously described (Edsjö et al., 2001), applied for three days from the day of plating, in the presence or virtual absence of non-neuronal cells. In control experiments, the culture medium was changed 3–4 h after cell plating in order to
Discussion
The present study provides the first evidence that NGF regulates synaptophysin (Syp), an integral protein of synaptic vesicles, in newborn TG neurons. In a virtual absence of non-neuronal cells, NGF increases the intensity of Syp immunoreactivity in neurites while decreasing the number of Syp-IR neuronal cell bodies identified by immunoreactivity for the pan-neuronal marker PGP 9.5.
Very little is known about effects of NGF on Syp expression. In pheochromocytoma cell line PC12, exogenous NGF
Acknowledgements
L.T. expresses her gratitude to Dr. J. Craig Baumgartner for support of this project, and to Jessica L. Martin for expert advice on digital image analysis. We thank Alexandra Brown and Matthew L. Shih for help with pilot experiments, Dr. Thomas Südhof for advice regarding the synaptophysin antibody, and Victoria Jenkins for a critical reading of the manuscript.
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2011, NeuroscienceCitation Excerpt :All procedures were approved by the Institutional Animal Care and Use Committee of the Oregon Health and Science University, and conformed to the Policies on the Use of Animals and Humans in Neuroscience Research approved by the Society for Neuroscience. Neuron-enriched TG cultures were prepared as previously described by our laboratory (Buldyrev et al., 2006; Tarsa and Balkowiec, 2009), and grown in Neurobasal-A medium (Invitrogen, Carlsbad, CA, USA) supplemented with B-27 (Invitrogen), 0.05 mg/ml penicillin-0.05 mg/ml streptomycin-0.1 mg/ml neomycin (Invitrogen), 0.5 mM l-glutamine (Invitrogen), and 2.5% fetal bovine serum (HyClone, Logan, UT, USA) for 4–6 days in 48-well tissue culture-treated plates (Nalge Nunc Int., Naperville, IL, USA) pre-coated with poly-d-lysine (0.1 mg/ml; Sigma, St. Louis, MO, USA) and laminin (0.4 μg/ml; Sigma; for quantitative RT-PCR and ELISA), or on poly-d-lysine/laminin-coated glass coverslips (for immunocytochemistry). The medium was replaced with fresh medium every 2–3 days.
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This work was funded by the Medical Research Foundation of Oregon and National Institutes of Health (HL076113) grants to A.B.