Brain-derived neurotrophic factor acutely inhibits AMPA-mediated currents in developing sensory relay neurons

Abstract

Brain-derived neurotrophic factor (BDNF) is expressed by many primary sensory neurons that no longer require neurotrophins for survival, indicating that BDNF may be used as a signaling molecule by the afferents themselves. Because many primary afferents also express glutamate, we investigated the possibility that BDNF modulates glutamatergic AMPA responses of newborn second-order sensory relay neurons. Perforated-patch, voltage-clamp recordings were made from dissociated neurons of the brainstem nucleus tractus solitarius (nTS), a region that receives massive primary afferent input from BDNF-containing neurons in the nodose and petrosal cranial sensory ganglia. Electrophysiological analysis was combined in some experiments with anterograde labeling of primary afferent terminals to specifically analyze responses of identified second-order neurons. Our data demonstrate that BDNF strongly inhibits AMPA-mediated currents in a large subset of nTS cells. Specifically, AMPA responses were either completely abolished or markedly inhibited by BDNF in 73% of postnatal day (P0) cells and in 82% of identified P5 second-order sensory relay neurons. This effect of BDNF is mimicked by NT-4, but not NGF, and blocked by the Trk tyrosine kinase inhibitor K252a, consistent with a requirement for TrkB receptor activation. Moreover, analysis of TrkB expression in culture revealed a close correlation between the percentage of nTS neurons in which BDNF inhibits AMPA currents and the percentage of neurons that exhibit TrkB immunoreactivity. These data document a previously undefined mechanism of acute modulation of AMPA responses by BDNF and indicate that BDNF may regulate glutamatergic transmission at primary afferent synapses.

Fig. 1.

Effects of BDNF on AMPA currents in P0 nTS neurons. A, Sample recording of BDNF effect on AMPA currents in a P0 nTS neuron. The neuron was first superfused with control bath solution followed by a 2 sec pulse of 150 μmAMPA alone (horizontal bar; AMPA). The solution was then switched to one containing BDNF (50 ng/ml), and, 1 min later, 150 μm AMPA plus 50 ng/ml BDNF was simultaneously applied for 2 sec (AMPA + BDNF). After 1 min rinse with control bath solution, the application of AMPA alone was repeated (AMPA recovery). Calibration: 4 sec, 50 pA.B, The distribution of AMPA currents in the presence (top panel) and absence (bottom panel) of BDNF in the entire population of P0 nTS neurons tested. AMPA currents in the presence of BDNF are expressed as a percentage of control AMPA currents evoked by the application of AMPA alone before BDNF treatment. AMPA currents in the absence of BDNF represent currents evoked by a second control application of AMPA expressed as a percentage of the first control application. The distribution of control AMPA currents shows a variability of 25% in the control responses. Therefore, the effect of BDNF was considered significant when the AMPA current in the presence of BDNF was <75% of the control AMPA current.

Fig. 2.

Comparison of the effects of three subsequent applications of AMPA + BDNF (closed circles) after control application of AMPA alone (open circle, time 0). Recovery from the BDNF effect was reached 1 min after BDNF was removed from the bath (open circle). n = 82; ** p < 0.01; *** p < 0.001.

Fig. 3.

Effects of BDNF on AMPA currents in P5/P9 identified second-order sensory neurons. A, Confocal image (a single optical section of 719 nm) of a P9 nTS neuron showing an attached, DiA-filled, synaptic bouton (arrow), taken 6 hr after dissociation. Scale bar, 5 μm. B,C, Mean integrated currents evoked by a control 2 sec application of AMPA alone (AMPA control), simultaneous application of AMPA and BDNF after 1 min BDNF pretreatment (AMPA + BDNF), and after return to superfusion with control bath solution (AMPA recovery), recorded in P5/P9 labeled nTS neurons (B; n = 9), and compared to P0 neurons (C; n= 82). * p < 0.05; **p < 0.01; *** p < 0.001.

Fig. 4.

The effects of BDNF on AMPA currents are TrkB-mediated. A, TrkB immunoreactivity in the commissural subnucleus of the nucleus tractus solitarius in a P0 rat (transverse section). Scale bar, 100 μm. In control sections in which the primary antibody was omitted during the staining procedure, no staining was detected above background (data not shown).B, TrkB-positive (large arrow) and TrkB-negative (small arrow) cell in a P0 nTS culture, 24 hr after plating. Scale bar, 20 μm. C, Sample recording of the AMPA currents measured in a P0 nTS neuron during control AMPA application (AMPA) and during the coapplication of AMPA and BDNF after 1 min BDNF pretreatment (AMPA + BDNF), before (Control) and after K252a treatment. The cell was superfused with 200 nm K252a for 12 min. Calibration: 4 sec, 50 pA. D, Mean integrated currents in P0 nTS neurons evoked by control 2 sec application of AMPA alone (AMPA control), AMPA, and BDNF after 1 min BDNF pretreatment (AMPA + BDNF), and after return to superfusion with control bath solution (AMPA recovery) in the absence or presence of 200 nm K252a; n= 8. E, Mean integrated currents in four P0 nTS neurons in which the effects of 200 nm K252b on BDNF inhibition of AMPA currents were tested. AMPA responses were abolished by BDNF in all neurons tested before and after K252b treatment.

Fig. 5.

The effects of BDNF, NT-4, and NGF on AMPA currents measured in P0 nTS neurons. A,B, The response to AMPA + BDNF was determined first, as described in Figure 1. After recovery of control AMPA responses, the response to AMPA + 50 ng/ml NT-4 (A) or 50 ng/ml NGF (B) was determined using the same protocol.A, n = 22; *p < 0.05; **p < 0.01; B,n = 12; *p < 0.05; n.s., not significant.