Elsevier

Neuropeptides

Volume 43, Issue 1, February 2009, Pages 47-52
Neuropeptides

Short communication
Nerve growth factor regulates synaptophysin expression in developing trigeminal ganglion neurons in vitro

https://doi.org/10.1016/j.npep.2008.09.009Get rights and content

Abstract

The role of neuronal growth factors in synaptic maturation of sensory neurons, including trigeminal ganglion (TG) neurons, remains poorly understood. Here, we show that nerve growth factor (NGF) regulates the intracellular distribution of the synaptic vesicle protein synaptophysin (Syp) in newborn rat TG neurons in vitro. While reducing the number of Syp-positive cell bodies, NGF dramatically increases Syp immunoreactivity in both proximal and distal segments of the neurite. Intriguingly, the increase in Syp immunoreactivity occurs only in neuron-enriched cultures, in which the number of non-neuronal cells is significantly reduced. Together, our data indicate that NGF is a candidate molecule involved in early postnatal maturation of TG neurons, including control of presynaptic assembly, and thereby formation of synaptic connections.

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.

Section snippets

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.

References (27)

  • H. Yang et al.

    Immunocytochemical evidence that most sensory neurons of the rat molar pulp express receptors for both glial cell line-derived neurotrophic factor and nerve growth factor

    Arch. Oral Biol.

    (2006)
  • C. Bandtlow et al.

    Oligodendrocytes arrest neurite growth by contact inhibition

    J. Neurosci.

    (1990)
  • M.A. Bruno et al.

    Long-lasting rescue of age-associated deficits in cognition and the CNS cholinergic phenotype by a partial agonist peptidomimetic ligand of TrkA

    J. Neurosci.

    (2004)
  • Cited by (14)

    • Neuritogenic effect of sea cucumber glucocerebrosides on NGF-induced PC12 cells via activation of the TrkA/CREB/BDNF signalling pathway

      2018, Journal of Functional Foods
      Citation Excerpt :

      As is well known, several marker proteins are involved in the NGF-dependent stimulation of sympathetic neurons as well as in sensory neuron survival and differentiation. For example, the expression of synaptophysin significantly increases during the differentiation of the trigeminal nerve (Tarsa & Balkowiec, 2009). Similar results have been obtained from the analysis of GAP-43 expression.

    • Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse

      2014, Journal of Nutritional Biochemistry
      Citation Excerpt :

      It has been known that the NGF-induced activation of transcription factors might cause neurite growth, synaptogenesis and memory formation [25,26,28]. NGF treatment has been also reported to dramatically increase the expression of synaptophysin, the most abundant integral presynaptic vesicle protein, in both neuron-enriched cultures and rat brain [28,44]. In the present study, GE administration significantly increased the expression levels of synaptophysin and PSD-95 in the mouse hippocampus (Fig. 7), suggesting enhancement of synapse formation mediated by elevated NGF levels.

    • Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo

      2011, Neurochemistry International
      Citation Excerpt :

      At the present stage of our work we did not attempt to systematically study possible synaptic currents of the engrafted DPSCs. In this regard, our present preliminary data showing synaptophysin, a protein synthetized during synapsis formation (Tarsa and Balkowiec, 2009), expression in implanted DPSCs indicates that these cells may be able to integrate into brain circuitries. According to previous data obtained using engrafted cells of embryonic stem cell origin, spontaneous maturation and synaptic integration was found to be suppressed in vivo (Demeter et al., 2004).

    • Tumor necrosis factor-α increases brain-derived neurotrophic factor expression in trigeminal ganglion neurons in an activity-dependent manner

      2011, Neuroscience
      Citation 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.

    View all citing articles on Scopus

    This work was funded by the Medical Research Foundation of Oregon and National Institutes of Health (HL076113) grants to A.B.

    View full text