Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation Yury Kovalchuk, Eric Hanse, Karl W. Kafitz, and Arthur Konnerth

Experiments were performed on dentate granule cells in hippocampal slices (300 m) from 28-45-day-old Balb/c mice (40). The animals were anaesthetized by exposure to CO₂, decapitated and the brains removed. Slices were incubated at 33°C in oxygenated standard solution for at least 40 - 60 min before they were transferred to the recording chamber. The standard solution contained (in mM): 125 NaCl, 2.5 or 3.5 KCl, 2 CaCl₂, 1.2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 20 glucose, 0.01 bicuculline, bubbled with 95% O₂ and 5% CO₂. Combined electrophysiological recordings and confocal Ca⁺² imaging were performed with an EPC9 patch-clamp amplifier and a rapid confocal laser-scanning system, respectively. The pipette solution contained (in mM): 140 K-gluconate, 10 NaCl, 4 Mg-ATP, 2 Na₂-ATP, 0.4 Na-GTP, 10 HEPES, 10 Phosphocreatine, 0.1 Oregon Green 488 BAPTA-1, pH 7.3. In some experiments 1-2 mM D890 was added to the pipette solution. The pipette resistance ranged from 5 to 8.5 MOhm and the series resistance (Rs) from 23 to 36 MOhm. Whole-cell recordings were performed at 30°C. Membrane potential was set to -75 to -80 mV. For synaptic stimulation, afferent fibers in the middle third of the molecular layer were activated by voltage pulses (5-18 V, 50-100 s duration) delivered through a glass pipette that was positioned extracellularly under visual control. BDNF stock solution was diluted in the standard solution to reach the final concentration of 20-50 ng/ml. BDNF was pressure ejected from the micropipettes by using a Picospritzer. The output pressure was set to 6-20 psi and the pulse duration to 3 ms - 600 ms. For local dendritic application the ejection pipette was positioned about 5-25 m away from the dendrite under the study.

Supplemental Figure 1. Actions of various neurotrophins in dentate granule cells. (a) BDNF-evoked current in a dentate granule cell in a hippocampal slice obtained from 5 weeks old mouse. (b-d) Current recordings in response to the application of NT-4/5, NT-3 and NGF, respectively. All recordings were obtained in the same cell. Similar results obtained in 4/4 different cells.

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Supplemental Figure 2. Block of BDNF-evoked currents by K252a. Reversible block of the BDNF-evoked inward current in a dentate granule cell by K252a. Similar results obtained in 4/4 cells. As for CA1 pyramidal cells (Kafitz et al., Nature, 1999), the incubation with K252a for >10 min was needed for the block to occur, while full washout was obtained after 15-20 min of perfusion with standard Ringer solution.

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Supplemental Figure 3. Papain-treated BDNF is ineffective. Left panel: BDNF-evoked current in a dentate granule cell. Right panel: Treatment of BDNF from the same batch as that used in the left panel for 15 min with 0.5 mg/ml, 200 U papain (Worthington, USA) produced no response. Similar results obtained in 4/4 cells.

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Supplemental Figure 4. BDNF-application in the presence of the BDNF scavenger TrkB-IgG. Incubation of the slice with TrkB-IgG (Regeneron) for more than 1 hour abolished the BDNF-evoked current in a dentate granule cell. Similar results obtained in 4/4 cells.

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Supplemental Figure 5. BDNF-mediated LTP in CA1 hippocampal pyramidal cells. (A) CA1 pyramidal cell (loaded with 100 M Oregon BAPTA-1) of a hippocampal slice obtained from a 4 weeks old mouse. (B) While weak stimulation alone (burst, 10 stimuli at 50 Hz producing subthreshold EPSPs) was ineffective, pairing of such a burst with a 600 ms BDNF pulse (50 ng/ml) produced, as in dentate granule cells, robust LTP. Pairing applied twice with an interval of 20 s. Similar results were obtained in 3/4 CA1 pyramidal cells.

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