Endogenous brain-derived neurotrophic factor triggers fast calcium transients at synapses in developing dendrites

Abstract

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of the formation of functional neuronal networks. BDNF signaling regulates neuronal development not only globally, at the level of entire neurons or networks, but also at a subcellular level and with high temporal specificity; however, the spatiotemporal characteristics of intrinsic BDNF signaling are essentially unknown. Here, we used calcium imaging to directly observe intrinsic BDNF signaling in developing hippocampal neurons. We found that blocking intrinsic BDNF signaling with function-blocking BDNF antibodies (alphaBDNF) or K252-a reduced the frequency of spontaneously occurring fast and localized calcium rises in dendrites. Conversely, focal application of BDNF evoked fast and local dendritic calcium transients, which required activation of TrkB (tropomyosin-related kinase B) receptors as well as activation of voltage-gated sodium and calcium channels. Virus-mediated expression of PSD-95:CFP (postsynaptic density-95 tagged with cyan fluorescent protein) revealed that spontaneous local calcium transients occurred frequently at postsynaptic sites along the dendrite. The frequency of synaptically localized calcium transients was specifically reduced by blocking intrinsic BDNF signaling, whereas nonsynaptic calcium rises were not affected. Furthermore, focal BDNF delivery evoked localized and fast calcium elevations specifically at postsynaptic sites. Together, our results demonstrate that BDNF-dependent calcium signaling in developing hippocampal neurons is fast and occurs at synapses. These temporal and spatial characteristics of intrinsic BDNF signaling as well as its relative abundance renders BDNF an ideal signaling molecule in the establishment of specific synaptic connectivity and functional neuronal networks.

Figure 1.

Frequency of spontaneously occurring local calcium transients in dendrites of developing hippocampal neurons is decreased by BDNF antibody or K252-a application. A, Electroporated single CA3 pyramidal neuron (P2 + 3 d in vitro) filled with Oregon Green BAPTA-1. Enlarged part of the dendrite shows spontaneous local increase in [Ca ²⁺]_i, represented as ΔF/F₀ in pseudocolor. Arrowheads indicate the extent of the measured dendritic segment used for pseudo line scan and trace. B, Western blot analysis of TrkB receptor and actin in hippocampal lysates from developing rats (P2). Both full-length TrkB and truncated TrkB (molecular weights of 145 and 95 kDa, respectively) were present in the hippocampus (HC) but absent in the control (lysate of HEK 293 cells). Actin served as the loading control. C, Frequency of spontaneous local calcium transients before, during, and after bath application of K252-a normalized to baseline frequency. Calcium activity is significantly reduced during K252-a application. n = 11 neurons; error bars indicate SEM (*p < 0.05). D, Similarly, bath application of αBDNF causes a significant and reversible decrease in the frequency of spontaneous local calcium transients. n = 11 neurons (*p < 0.05). E, The frequency of spontaneous global calcium transients is unchanged during BDNF antibody application.

Figure 2.

BDNF bath application causes a strong increase in the frequency of global calcium transients. A, Spontaneous [Ca ²⁺]_i changes in the dendritic segment marked with the arrowheads are shown in pseudo line scans as ΔF/F₀. Line scans are shown before and during BDNF bath application (at 3 different time points) and after washout. B, The frequency of spontaneously occurring global calcium transients is significantly (*p < 0.05) increased during bath application of BDNF (n = 8 neurons, values normalized to baseline frequency). C, Frequency of spontaneous local calcium transients is not significantly increased during BDNF bath application (n = 12 neurons). D, Bolus loading of CA3 neurons in control slices shows an acute increase in [Ca ²⁺]_i during BDNF bath application and a delayed increase in correlated network activity (traces correspond to the encircled neurons in the left image). E, Neurons in slices incubated for 12 h in TeTX also reveal an immediate increase in [Ca ²⁺]_i to BDNF bath application but otherwise remain silenced. F, Frequency of global calcium transients in control- and TeTX-incubated slices before and after BDNF bath application (80 neurons from 8 slices; control slices: before, 0.8 ± 0.3; after, 4.9 ± 0.8 global calcium transients per minute; TeTX-incubated slices: before, 0.07 ± 0.02; after, 0.17 ± 0.06 global calcium transients minute; *p < 0.05). G, Averaged acute calcium responses to fast BDNF bath application in control slices and in TeTX-incubated slices (80 neurons from 8 slices).

Figure 3.

Dendritic calcium transients can be elicited by focal BDNF application and are dependent on activation of TrkB receptors as well as voltage-gated sodium and calcium channels. A, An electroporated CA3 neuron with the BDNF application pipette, placed ∼20 μm from the dendrite (top right), responds to a single 40 ms BDNF pulse with a strong local increase in [Ca ²⁺]_i. B, Left trace shows an average of calcium responses to six BDNF pulses applied at the indicated dendritic region in A. Right trace shows averaged responses to six pulses of a control solution (heat-inactivated BDNF) in another neuron. C, Pharmacology of BDNF-evoked calcium transients: amplitudes of BDNF-evoked calcium transients are shown as percentage of baseline. Bath application of K252-a, TTX, and CdCl₂ reduced the amplitude of the calcium transients evoked by local BDNF application significantly (*p < 0.05). In contrast, no reduction in the amplitude of the BDNF-induced calcium transients was observed during bath application of CPA, SKF 96365 hydrochloride, or in TeTX incubated slices.

Figure 4.

BDNF-mediated local calcium transients occur preferentially at synaptic sites. A, Neuron expressing PSD-95:CFP (shown in red) by virus-mediated gene transfer after immunolabeling with anti-synapsin antibodies and high-resolution confocal reconstruction (left, maximum intensity projection of the neuron in red, single plane of synapsin staining in green). Arrow indicates a synapse as PSD-95:CFP and synapsin overlap. High-magnification (x–y) and orthogonal (y–z) views of the marked synapse (yellow pixels in both views). B, PSD-95:CFP (blue) expressing hippocampal neuron electroporated with the calcium indicator Oregon Green BAPTA-1 (OGB) (green). Enlarged part of the dendrite shows PSD-95:CFP puncta; the puncta are also revealed in a ratio plot of CFP/Oregon Green BAPTA-1 pixel intensity along the dendrite. Line scan of the enlarged part of the dendrite shows spontaneously occurring local calcium transients at four of the PSD-95:CFP sites (right). C, Distribution of spontaneous local calcium transients with respect to putative synaptic sites (99 local calcium signals from 8 neurons). Most transients occur at or very close to the nearest synapse (the mean intersynapse distance of 17.3 ± 1.9 μm agrees with numbers observed in CA1 neurons in the first postnatal week) (Steward and Falk, 1991). D, Frequency of spontaneous local calcium transients occurring at a synapse (<2 μm apart) and at nonsynaptic sites (>2 μm apart from the nearest synapse) before, during, and after bath application of BDNF antibodies. The frequency of local calcium transients at synaptic sites, but not at nonsynaptic sites, is significantly reduced during bath application of BDNF antibodies (n = 8 neurons; percentage of baseline; *p < 0.05).

Figure 5.

BDNF-evoked local calcium transients can be induced at synaptic sites. A, PSD-95:CFP-expressing pyramidal neuron, electroporated with Oregon Green BAPTA-1 (OGB-1) (left); arrowheads mark PSD-95:CFP puncta along the apical dendrite. A BDNF pulse caused a strong calcium rise (represented as ΔF/F₀ values) exactly at the sites of two putative synapses (right). Bottom, Line scan reveals that calcium transient induced by the BDNF pulse (red arrow) starts at the two PSD-95:CFP sites and shows exactly there the highest fluorescence increase. B, Another example of a BDNF-evoked calcium transient at a PSD-95:CFP site. C, BDNF-evoked calcium transients at a PSD-95:CFP punctum (blue trace) and at an adjacent region (gray trace) indicated in B. D, Maximal amplitudes of ΔF/F₀ within 1 s after the BDNF pulse at nonsynaptic dendritic sites and at PSD-95:CFP puncta (*p < 0.05; n = 9).