A caged Ab reveals an immediate/instructive effect of BDNF during hippocampal synaptic potentiation

Abstract

Neurotrophins have been shown to be involved in functional strengthening of central nervous system synapses. Although their general importance in this process is undisputed, it remains unresolved whether neurotrophins are truly mediators of synaptic strengthening or merely important cofactors. To address this question, we have devised a method to inactivate endogenous brain-derived neurotrophic factor (BDNF) with high time resolution by "caging" a function-blocking mAb against BDNF with a photosensitive protecting compound. Different assays were used to show that this inactivation of the Ab is reversible by UV light. Synaptic potentiation after theta-burst [corrected] stimulation in the CA1 region of acute hippocampal slices was significantly less when applying the unmodified Ab compared with the caged Ab. Importantly, photoactivation of the caged Ab during the time of induction of synaptic enhancement led to a marked decrease in potentiation. Our experiments therefore strengthen the view that endogenous BDNF has fast effects during induction of synaptic plasticity. The results additionally show that caged Abs can provide a tool for precise spatiotemporal control over endogenous protein levels.

Figure 1

Reversible inactivation of a function-blocking mAb against the neurotrophin BDNF. (A) Illustration of the caging reaction with NVOC-Cl: free amino groups of lysines on the Ab are modified by reaction with the amine-reactive caging compound. A chemically stable but photolabile carbamate is formed when the nitrogen of a free amine attacks the carbonyl carbon of NVOC-Cl to displace the chloride. Irradiation causes an internal redox reaction to generate a nitrosobenzyl and an unstable carbamic acid which spontaneously decarboxylates to regenerate the free amine. (B) ELISA demonstrating the reduced binding of the Ab to BDNF after modification with the caging group and its successful reactivation by UV light. The remaining signal of the caged Ab (21%) reflects unspecific binding (see text). Filled bars show the binding activity of the unirradiated Ab; open bars show the activity after irradiation. (C) Bioassay to test the blocking activity of the caged or uncaged Ab. (Bar = 100 μm.) (Left) Chick nodose ganglion neurons cultured in the presence of BDNF and caged Ab. (Right) Parallel preparation after adding the irradiated Ab. (D) Quantification of the altered function-blocking properties. Filled bars: percentage of surviving cells with unirradiated Ab; open bars, percentage of surviving cells after adding the irradiated Ab.

Figure 2

Photoactivation in hippocampal slices. (A) Illustration of the setup for the physiological experiments. The irradiation spot (produced with a 32× objective) was placed in the CA1 area. (Bar = 750 μm.) (B–E) Diffusion of the Ab in slices assessed by NVOC-caged Rhodamine green coupled to BDNF Abs. The photoactivation paradigm was identical to that used in the LTP experiments. (B–D) Examples of photoactivation within the slice. (B) Area of irradiation before photoactivation, (C) immediately after irradiation, and (D) 60 min after irradiation. (Bar = 125 μm.) (E) Average time course of relative fluorescence within the photoactivated spot. The slow decrease in fluorescence over time shows the decrease in concentration of photoactivated molecules caused by diffusion. Fluorescence intensity was measured in 10-min intervals (●, n = 7) or only once after 60 min (□, n = 7) to demonstrate that bleaching is not the cause of the decay in fluorescence.

Figure 3

LTP in the CA1–Schaffer collateral pathway of hippocampal slices in the presence of caged (▴) or unmodified (□) BDNF-Ab (concentration 2 μg/ml). After TBS (indicated by the ▾ symbol), synaptic potentiation was significantly larger in slices treated with caged Ab compared with unmodified Ab. Inset depicts two EPSP traces for the caged Ab experiment, 10 min before (thin trace) and 60 min after (thick trace) TBS.

Figure 4

Effect of photoactivation of the Ab during the time of TBS. Irradiation was started 2 min before and stopped 2 min after TBS (indicated by the dotted line). (A) Uncaging of the BDNF Ab (□) significantly reduced the degree of synaptic potentiation up to 50 min after TBS compared with experiments without irradiation (▴). (B) Control experiments show no significant difference between uncaged (□) and caged (▴) BSA.

Figure 5

Experiments similar to those in Fig. 4, with photoactivation starting only 7 s before TBS. In this case, a modified c-myc Ab (against the human c-myc 9E10 epitope) was used as control. Uncaging of the BDNF-Ab (□) reduced potentiation during the first 15 min compared with the control group (▴).