Nerve growth factor rapidly induces prolonged acetylcholine release from cultured basal forebrain neurons: differentiation between neuromodulatory and neurotrophic influences

Abstract

Long-term exposure to nerve growth factor (NGF) is well established to have neurotrophic effects on basal forebrain cholinergic neurons, but its potential actions as a fast-acting neuromodulator are not as well understood. We report that NGF (0.1-100 ng/ml) rapidly (<60 min) and robustly enhanced constitutive acetylcholine (ACh) release (148-384% of control) from basal forebrain cultures without immediate persistent increases in choline acetyltransferase activity. More ACh was released in response to NGF when exposure was coupled with a higher depolarization level, suggesting activity dependence. In a long-term potentiation-like manner, brief NGF exposure (10 ng/ml; 60 min) induced robust and prolonged increases in ACh release, a capacity that was shared with the other neurotrophins. K252a (10-100 nm), BAPTA-AM (25 microm), and Cd(2+) (200 microm) prevented NGF enhancement of ACh release, suggesting the involvement of TrkA receptors, Ca(2+), and voltage-gated Ca(2+) channels, respectively. Forskolin (10 microm), a cAMP generator, enhanced constitutive ACh release but did not interact synergistically with NGF. Tetrodotoxin (1 microm) and cycloheximide (2 microm) did not prevent NGF-induced ACh release, indicative of action at the level of the cholinergic nerve terminal and that new protein synthesis is not required for this neurotransmitter-like effect, respectively. In contrast, after a 24 hr NGF treatment, distinct protein synthesis-dependent and independent effects on choline acetyltransferase activity and ACh release were observed. These results indicate that neuromodulator/neurotransmitter-like (protein synthesis-independent) and neurotrophic (translation-dependent) actions likely make distinct contributions to the enhancement of cholinergic activity by NGF.

Fig. 1.

Activity-dependent enhancement of ACh release by NGF. Cultures were preexposed to NGF (10 ng/ml) for 60 min in low K⁺ (6 mm) buffer. ACh release was then evaluated from low-activity (6 mm K⁺) or high-depolarization (25 mm K⁺) conditions for a 15 min period. Columns represent increased ACh (femtomoles per well per minute ± SEM;n = 8) associated with NGF preexposure versus the same depolarizing conditions without NGF preexposure. Significance was determined using Student's t test (*p < 0.001 vs 6 mmK⁺).

Fig. 2.

NGF acutely enhances constitutive ACh release from embryonic basal forebrain neurons in a concentration-dependent manner during a short-term exposure but does not induce persistent ChAT activity changes. Data are expressed as a percentage of release or ChAT activity in the absence of NGF [mean ± SEM; 0 ng/ml (n = 59), 0.1 ng/ml (14), 0.5 ng/ml (14), 1 ng/ml (37), 10 ng/ml (27), and 100 ng/ml (19); control ACh release was ∼630 fmol/well for the 45 min exposure period, representing ∼14 fmol per well per minute]. Significance was determined using a one-way ANOVA with Tukey's post-test (*p < 0.001 vs control).

Fig. 3.

K252a (10–100 nm) prevents NGF (10 ng/ml) enhancement of ACh release during a short-term exposure. Data are expressed as a percentage of release in the absence of NGF and K252a (mean ± SEM; n = 6–8). Statistical analysis was performed using a two-way ANOVA with Tukey's post-test (*p < 0.001 vs K252a only, at the same concentration; ^†p = 0.0627 vs cultures receiving neither K252a nor NGF).

Fig. 4.

Forskolin (10 μm) alone increases ACh release but does not synergistically enhance NGF (1 ng/ml)-induced ACh release during a short-term exposure in low K⁺(6 mm) conditions. Data are expressed as a percentage of release in the absence of forskolin and NGF (mean ± SEM;n = 6). Significance was determined using a one-way ANOVA with Tukey's post-test (*p < 0.001 vs control; ^†p < 0.001 vs both NGF and forskolin).

Fig. 5.

NGF (1 ng/ml)-enhanced ACh release involves Ca²⁺. A, The intracellular Ca²⁺ chelator BAPTA-AM (25 μm) inhibits NGF-induced ACh release during a short-term exposure. Data are expressed as a percentage of release in the absence of BAPTA-AM and NGF (mean ± SEM). Significance was determined using a one-way ANOVA with Tukey's post-test (*p < 0.001 vs control;^† p < 0.001 vs NGF/BAPTA-AM).B, NGF-induced ACh release is inhibited by the voltage-gated Ca²⁺ channel antagonist Cd²⁺ (200 μm). Data are expressed as a percentage of release in the absence of Cd²⁺ and NGF (mean ± SEM; n = 6). Significance was determined using a one-way ANOVA with Tukey's post-test (*p < 0.001 vs control;^†p < 0.001 vs NGF/Cd²⁺).

Fig. 6.

Brief exposure to NGF induces prolonged increase in ACh release. A, NGF (10 ng/ml for 60 min) treatment is associated with increased ACh release for at least 4 hr. Data are normalized according to release from control wells at the same hour and are expressed as mean ± SEM (n = 17–23). Statistical analysis was performed using repeated measure one-way ANOVA with Tukey's post-test; *p < 0.001 vs control.B, Treatment with NGF (10 ng/ml for 60 min) was associated with enhanced ACh release during the subsequent 60 min and was specific to availability during the defined exposure period, because a p75NTR-IgG fusion protein (5 μg/ml) only blocked the effect when coadministered with NGF. Data are normalized according to control wells and are expressed as mean ± SEM (n = 4–8). Statistical analysis was performed using a one-way ANOVA with Tukey's post-test: *p < 0.001 vs control,^†p < 0.001 vs NGF/co-p75NTR-IgG fusion protein.

Fig. 7.

Evidence for protein synthesis-dependent and -independent effects of NGF on ChAT activity and ACh release, respectively. ChAT activity (A) and constitutive ACh release (B) were compared after 6, 12, and 24 hr exposures to NGF (100 ng/ml) and/or cycloheximide (CY; 2 μm). Data are normalized according to controls and are expressed as mean ± SEM (6 hr,n = 4; 12 hr, n = 4–8; 24 hr,n = 20–24). Statistical analysis was performed using two-way ANOVAs with Tukey's post-test: *p <0.05 vs vehicle-treated control and ^†p<0.05 vs NGF/CY (within ChAT and ACh, same hour);^‡p < 0.05 vs NGF at 6 and 12 hr (within ChAT and ACh); ^§p< 0.05 vs NGF/CY at 6 and 12 hr (within ACh). Furthermore, at 6, 12, and 24 hr, the percentage changes in ACh release and ChAT activity were different within NGF and NGF/CY groups (p < 0.05).