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

Abstract

The neurotrophin (NT) brain-derived neurotrophic factor (BDNF) plays an essential role in the formation of long-term potentiation (LTP). Here, we address whether this modulation by BDNF requires its continuous presence, or whether a local increase in BDNF is necessary during a specific time period of LTP initiation. Using electrical field stimulation of primary cultures of hippocampal neurons, we demonstrate that short high-frequency bursts of stimuli that induce LTP evoke also an instantaneous secretion of BDNF. In contrast, stimuli at low frequencies, inducing long-term depression, do not enhance BDNF secretion, suggesting that BDNF is specifically present, and thus required, at the time of LTP induction. The field-stimulation-mediated BDNF secretion depends on the formation of action potentials and is induced by IP(3)-mediated Ca(2+) release from intracellular stores. Experiments, aimed at determining the sites of NT secretion that use NT6, showed similar patterns of surface labeling by field stimulation to those shown previously by high potassium.

Figure 1

BDNF secretion in response to different stimulation frequencies. (A) Field stimulation setup: a culture of hippocampal neurons is placed between parallel oriented platinum wires, which are connected to a stimulus isolator and a pulse generator. (B) Current clamp recordings were performed in hippocampal pyramidal neurons in response to 50-Hz stimulus trains and 50-Hz burst patterns. Each stimulus (stimulus artifact, ▴) is followed by one AP (Δ). (C) Trains of 600 pulses at 50 Hz (n = 52) but not at 10 Hz (n = 37), elicited a significant BDNF secretion. (D) BDNF secretion in response to a train of 300 pulses at 50 Hz (n = 19) was comparable to secretion evoked by a 50-Hz burst pattern (n = 17). Stimuli (300 pulses) at 100 Hz (applied as bursts or trains, n = 36) did not further enhance BDNF secretion as compared with trains or bursts at 50 Hz (n = 36). The difference (in C and D) of secretion levels induced by 50 Hz results from the fact that experiments were performed independently with different cell preparations. Fractions were collected over 5 min. Values represent the mean ± SE. n.s., Not significant (P > 0.05); **, P < 0.01.

Figure 2

BDNF secretion and LTP are triggered by the same stimulation patterns, whereas LTD-inducing patterns do not induce BDNF secretion. BDNF secretion in response to different LTP- and LTD-inducing stimulation patterns was measured over a time period of 5 min. Numbers of stimuli (300) within the first three columns were identical: 3 × 100 (three trains of 100 stimuli at 100 Hz, interburst interval of 20 s; n = 38), 7× TBS (one TBS consists of ten bursts each of four pulses at 100 Hz, with an interburst interval of 200 ms, inter-TBS interval 10 s; n = 46), and 1 Hz (train of 300 pulses at 1 Hz; n = 18). Also, weaker LTP-inducing stimuli (3 × 30: three bursts each of 30 pulses at 100 Hz, interburst interval 20 s; 3× TBS) are sufficient to evoke a substantial BDNF secretion (n = 22). Values given represent the mean ± SE. n.s., Not significant (P > 0.05); **, P < 0.01.

Figure 3

Short bursts of stimuli are sufficient to evoke an instantaneous BDNF secretion. (A) Hippocampal neurons were stimulated for 1–5 s (50–250 pulses) with trains of stimuli at 50 Hz. BDNF concentrations of supernatants taken in 1-min intervals were measured. A pulse of 2 s was sufficient to stimulate BDNF secretion. Values given represent the mean ± SE. n.s., Not significant (P > 0.05); **, P < 0.01; *, P < 0.05. (1–3 s, n = 11; 5 s, n = 28). (B) Hippocampal neurons were stimulated either with 100 Hz tetani (three trains of 100 stimuli at 100 Hz, interburst interval of 20 s; n = 11), 7 TBS (n = 13), or trains of 250 pulses at 50 Hz (n = 6). Supernatants were collected each minute. Values are normalized to the average of the first two basal values and are represented as the mean ± SE. The fat line represents the mean of all experiments (n = 30). All experiments show a similar time course: a constant baseline and immediate BDNF secretion in the fraction collected during the stimulus.

Figure 4

AP firing is required for regulated BDNF secretion. (A) BDNF secretion in response to trains of 250 pulses at 50 Hz in absence (n = 17) and presence (n = 22) of 1 μM TTX. BDNF secretion measured in supernatants collected over 5 min was completely abolished if APs were blocked. Values given represent the mean ± SE. n.s., Not significant (P > 0.05). (B) Patch clamp recordings in current clamp mode of hippocampal neurons under the same conditions as in A. TTX (1 μM) abolished the formation of APs in response to field stimulation. One stimulus in presence and in absence of TTX is enlarged.

Figure 5

Electrical field stimulation-induced BDNF secretion depends on Ca²⁺ mobilization from intracellular stores. In the first set of experiments Ca²⁺ stores were depleted by preincubation with caffeine (10 mM) and thapsigargin (10 μM) for 20 min. This pretreatment abolished subsequent BDNF secretion by 50 Hz field stimulation (n = 33). In a second experiment hippocampal neurons were preincubated (2–20 min) with 75 μM 2-APB, an inhibitor of IP₃ receptor function. 2-ABP blocked BDNF secretion induced by 50 Hz field stimulation (n = 42). Values given represent the mean ± SE. n.s., Not significant (P > 0.05); **, P < 0.01.

Figure 6

Sites of field-stimulation-induced NT6myc secretion: (A) Representative examples of NT6myc surface distribution at the ultrastructural level after field stimulation with TBS or 50-Hz trains. Surface bound NT6myc was fixed immediately after secretion and detected via its myc tag, using preembedding immunogold cytochemistry (4 nm gold) at the neuronal surface. Secretion occurs from dendrites and axons and is not restricted to synaptic sites. Arrowheads point to secretion sites. a, axon; d, dendrite. (Scale bar, 100 nm.) (B) Quantitative evaluation of the NT6myc secretion at the electron microscopic level by stereological procedures. Pooled data from three independent experiments are presented. Values given represent the mean ± SE of three independent experiments. **, Statistically significant differences (gold particle per μm; P < 0,01) in comparison with stimulation with 10 Hz or 50 Hz in the presence of 1 μM TTX.