Characterization of Nerve Growth Factor (NGF) Release from Hippocampal Neurons: Evidence for a Constitutive and an Unconventional Sodium‐dependent Regulated Pathway

Abstract

We investigated the mechanism of neuronal nerve growth factor (NGF) release with regard to the potential function of NGF as a mediator of neuronal plasticity in the CNS. The analysis was performed in hippocampal slices and in primary cultures of hippocampal neurons, transiently transfected with an NGF cDNA construct to increase the level of NGF expression. In both systems there was activity-dependent NGF release initiated by high potassium (KCl), veratridine, glutamate or carbachol. Replacement of 90% of sodium in the medium with N-methyl-glucamine strongly reduced this release. The KCl- and veratridine-initiated NGF release was suppressed by tetrodotoxin; release by glutamate was less sensitive to tetrodotoxin but was sodium-dependent. The glutamate effect could be inhibited by GYKI52644, an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, but not by MK-801, an antagonist of NMDA receptors. The activity-dependent release of NGF did not depend on extracellular Ca2+, but was sensitive to the intracellular Ca2+ chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, and to depletion of intracellular calcium stores. Conversely, mobilization of Ca2+ from intracellular stores with caffeine and thapsigargin mimicked the effect of depolarization. Basal NGF release could be reduced by either temperature block (15 degrees C) or tetrodotoxin to approximately 50%. The combination of both treatments reduced NGF release to below the detection limit, suggesting that basal release has constitutive and regulated components, the latter presumably resulting from spontaneous activity of interconnected neurons.

Neurotrophin secretion from hippocampal neurons evoked by long-term-potentiation-inducing electrical stimulation patterns

“…4B) as such, is responsible for BDNF secretion. This interpretation is supported by the observation that KCl-evoked NT secretion was abolished in the presence of TTX (27), where neurons remained depolarized (Ϫ10 mV; see Fig. 9A, which is published as supporting information on the PNAS web site).…”

“…In previous studies analyzing regulated NT secretion, very long stimuli (5-60 min) were generally used for technical reasons [dead volume of the perfusion system, bath application of stimulating agents (19,24,27)] or to reach detectable levels of secreted BDNF (18,28). Electrical stimulation allowed us to determine the minimal number of stimuli necessary to reliably evoke NT secretion.…”

Single-Cell Characterization of Retrograde Signaling by Brain-Derived Neurotrophic Factor

“…It has been shown that this depolarizing GABAergic input can be critical for normal neuronal development (Borodinsky et al, 2003;Ge et al, 2006), and a possible mediator of this effect could be regulated release of BDNF, the expression of which has been shown to be enhanced by GABA application (Berninger et al, 1995). Although it has been reported that depolarization-induced release of BDNF is essentially eliminated by exposure to TTX (Blochl and Thoenen, 1995;Katz, 2000, 2002;Kojima et al, 2001;Gartner and Staiger, 2002) and would therefore not be expected to occur in response to moderate sustained depolarization, it is possible that the methodology used was not sensitive enough to detect it. We therefore chose to hold cells at Ϫ40 mV and use a change in presynaptic responsiveness as our assay.…”

Cellular Mechanisms Regulating Activity-Dependent Release of Native Brain-Derived Neurotrophic Factor from Hippocampal Neurons

“…To establish whether calcium release from caffeine-ryanodinesensitive stores is capable of inducing release of native BDNF from hippocampal neurons, we initially examined the effects of caffeine alone. Thirty minute treatment of hippocampal cultures with 30 M caffeine, an agonist of ryanodine receptors, resulted in a significant increase in BDNF release that was blocked by 10 min pretreatment with 50 M dantrolene, a ryanodine receptor antagonist (Blöchl and Thoenen, 1995) and 10 M thapsigargin (Fig. 6 A).…”

“…Previous studies have indicated that regulated secretion of neurotrophins requires calcium release from intracellular calcium stores, including caffeine-ryanodine-sensitive stores (Blöchl and Thoenen, 1995;Griesbeck et al, 1999;Canossa et al, 2001). To establish whether calcium release from caffeine-ryanodinesensitive stores is capable of inducing release of native BDNF from hippocampal neurons, we initially examined the effects of caffeine alone.…”