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The Journal of Neuroscience, October 1, 2000, 20(19):7417-7423
Activity-Dependent Release of Endogenous Brain-Derived
Neurotrophic Factor from Primary Sensory Neurons Detected by ELISA
In Situ
Agnieszka
Balkowiec and
David M.
Katz
Department of Neurosciences, Case Western Reserve University School
of Medicine, Cleveland, Ohio 44106
To define activity-dependent release of endogenous brain-derived
neurotrophic factor (BDNF), we developed an in vitro
model using primary sensory neurons and a modified ELISA, termed ELISA in situ. Dissociate cultures of nodose-petrosal ganglion
cells from newborn rats were grown in wells precoated with anti-BDNF antibody to capture released BDNF, which was subsequently detected using conventional ELISA. Conventional ELISA alone was unable to detect
any increase in BDNF concentration above control values following
chronic depolarization with 40 mM KCl for 72 hr. However, ELISA in situ demonstrated a highly significant increase
in BDNF release, from 65 pg/ml in control to 228 pg/ml in KCl-treated cultures. The efficacy of the in situ assay appears to
be related primarily to rapid capture of released BDNF that prevents
BDNF binding to the cultured cells. We therefore used this approach to
compare BDNF release from cultures exposed for 30 min to either continuous depolarization with elevated KCl or patterned electrical field stimulation (50 biphasic rectangular pulses of 25 msec, at 20 Hz,
every 5 sec). Short-term KCl depolarization was completely ineffective
at evoking any detectable release of BDNF, whereas patterned electrical
stimulation increased extracellular BDNF levels by 20-fold. In
addition, the magnitude of BDNF release was dependent on stimulus
pattern, with high-frequency bursts being most effective. These data
indicate that the optimal stimulus profile for BDNF release resembles
that of other neuroactive peptides. Moreover, our findings demonstrate
that BDNF release can encode temporal features of presynaptic neuronal activity.
Key words:
BDNF release; chronic depolarization; electrical field
stimulation; ELISA; ELISA in situ; frequency; patterned
stimulation; P-CREB; primary sensory neurons
Copyright © 2000 Society for Neuroscience 0270-6474/00/20197417-07$05.00/0
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