Opposing Roles for Endogenous BDNF and NT-3 in Regulating Cortical Dendritic Growth  A.Kimberley McAllister, Lawrence C. Katz, Donald C. Lo  Neuron  Volume 18, Issue 5, Pages 767-778 (May 1997) DOI: 10.1016/S0896-6273(00)80316-5

Figure 1 Exogenous BDNF and NT-3 Have Reciprocal Actions on Dendritic Growth in Layers 4 and 6 of Developing Visual Cortex Changes in dendritic complexity of pyramidal neurons from layers 4 and 6 are summarized for visual cortical slices treated for 36 hr with 200 ng/ml BDNF and NT-3 (see alsoMcAllister et al. 1995. Dendritic complexity is described in terms of a dendrite modification index (DMI; see Experimental Procedures): a zero value on these graphs indicates no statistically significant difference from untreated neurons, an increase in DMI reflects an increase in dendritic complexity, and a decrease in DMI reflects a decrease in dendritic complexity. (A) Exogenous BDNF stimulates dendritic growth by almost doubling the complexity of basal dendrites of layer 4 neurons, while exogenous NT-3 has essentially no effect. (B) In contrast, growth of layer 6 basal dendrites is stimulated by NT-3 but inhibited by BDNF. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 2 Opposite Effects of Endogenous BDNF and NT-3 on Dendritic Growth of Layer 4 Neurons (A) Endogenous neurotrophins are required for normal dendritic development in cortical pyramidal neurons. Representative camera lucida reconstructions of neurons from untreated slices and slices treated with each of the Trk receptor bodies (20 μg/ml) are shown. Neutralizing endogenous BDNF and NT-4/5 with TrkB–IgG (upper right) caused pronounced decreases in the growth of basal dendrites, while neutralizing NGF with TrkA–IgG (lower left) had no significant effect compared to untreated neurons (upper left). Surprisingly, neutralizing NT-3 with TrkC–IgG dramatically enhanced dendritic arbors (lower right). Blocking endogenous neurotrophins had no substantial effect on apical dendritic growth. Scale bar, 30 μm. (B) Opposite effects of endogenous BDNF and NT-3 on dendritic growth. Changes in dendritic complexity caused by treatment with each of the receptor bodies (20 μg/ml) for basal (A) and apical (B) dendrites of pyramidal neurons in layer 4. Left graph: neutralizing endogenous BDNF with TrkB–IgG decreased dendritic complexity by over 50% compared with untreated neurons, while neutralizing endogenous NGF with TrkA–IgG had no effect on dendritic growth. Neutralizing endogenous NT-3 with TrkC–IgG markedly increased dendritic complexity, by almost twofold. Right graph: neutralizing endogenous neurotrophins had little effect on growth of apical dendrites. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 3 Neutralizing Endogenous BDNF Results in Retraction of Layer 4 Basal Dendrites Dendritic complexity was decreased by 36 hr of treatment with 20 μg/ml TrkB–IgG compared with the original morphology of neurons at the start of culture, indicating that retraction of layer 4 basal dendrites occurs when endogenous BDNF is neutralized. Dendritic morphology at the beginning of the treatment period was assessed by fixing slices immediately following cutting and filling neurons intracellularly with Lucifer Yellow (seeMcAllister et al. 1995. Mean values ± SEM are plotted for the parameters of dendritic growth indicated. Neutralizing endogenous BDNF clearly decreased the total amount of basal dendrite and the number of primary dendrites, decreasing dendritic complexity overall by 49% in terms of the DMI. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 4 Penetration and Specificity of Trk Receptor Bodies (A) Trk receptor bodies penetrate cultured slices and remain intact. Trk–IgGs were recovered from cultured slices treated for 36 hr with 20 μg/ml of each receptor body by homogenization and extraction of Trk–IgGs with Protein-G agarose, as described in the Experimental Procedures. Recovered proteins were separated by SDS-PAGE and immunoblotted with an anti-human IgG antibody to visualize Trk–IgGs. The upper band with apparent molecular weight of 92 kDa corresponds to the Trk–IgGs and is not present in homogenates of untreated (UT) slices; the lower band at ∼50 kDa is non-specific and is present in all slices regardless of treatment. The similarity of recovery of TrkA–IgG, TrkB–IgG, and TrkC–IgG indicated that these receptor bodies penetrated slices to equivalent degrees and remained intact during the treatment period. No proteolytic fragments containing the human IgG Fc domain of the receptor body were observed. (B) TrkB–IgG, but not TrkA–IgG or TrkC–IgG, inhibits the effects of exogenous BDNF. The specificity of each of the receptor bodies was assayed by their ability to inhibit the growth-promoting effects of exogenous BDNF on layer 4 neurons. TrkB–IgG showed dose- dependent inhibition of the effects of 200 ng/ml of BDNF, with complete inhibition occurring at an equimolar equivalent of TrkB–IgG (2 μg/ml, shown in bold, left); a 10-fold lower concentration of TrkB–IgG had no inhibitory effect. In contrast, even at 10-fold molar excess (22 μg/ml), neither TrkA–IgG nor TrkC–IgG inhibited the dendritic growth stimulated by exogenous BDNF (right). Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 5 Neutralizing Endogenous NT-3 and Adding Exogenous BDNF Cause Similar Dendritic Enhancement Camera lucida reconstructions of the basal dendritic arborizations of layer 4 neurons from slices treated with either TrkC–IgG (20 μg/ml) or BDNF (200 ng/ml). Neurons depicted for each treatment represent the range of morphologies across the sampled populations. Neutralizing endogenous NT-3 elicited elongation and sprouting of new primary dendrites, similar to that induced by exogenous BDNF. This suggested that blocking endogenous NT-3 releases endogenous BDNF from inhibition, allowing endogenous BDNF to exert its effects on dendritic growth. Scale bar, 20 μm. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 6 TrkB–IgG Blocks the Dendritic Growth Induced by TrkC–IgG in Layer 4 Neurons (A) Camera lucida reconstructions of basal dendritic arbors for layer 4 neurons from control slices and slices treated with either TrkC–IgG, TrkB–IgG, or both Trk–IgGs together (20 μg/ml each). Basal dendritic arbors of neurons treated with both TrkB–IgG and TrkC–IgG were clearly reduced in complexity compared with TrkC–IgG-treated cells; they were also reduced compared with untreated neurons. These arbors resembled those of TrkB–IgG-treated neurons. Scale bar, 20 μm. (B) Blocking endogenous TrkB ligands prevents dendritic growth caused by neutralizing endogenous NT-3. Changes in dendritic complexity (DMI) for neurons from slices treated with either TrkB–IgG alone, TrkC–IgG alone, or both Trk–IgGs together. Blocking endogenous TrkB ligands with TrkB–IgG completely prevented the increase in dendritic complexity caused by neutralizing endogenous NT-3 with TrkC–IgG. Treatment with both Trk–IgGs caused dendritic retraction compared with untreated neurons, but not to the full extent caused by treatment with TrkB–IgG alone. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 7 Exogenous NT-3 Blocks Dendritic Growth Elicited by Exogenous BDNF Camera lucida reconstructions of basal dendritic arbors of layer 4 neurons from untreated slices and slices treated with either BDNF (200 ng/ml), NT-3 (200 ng/ml), or both neurotrophins together. BDNF treatment caused striking growth of basal dendrites while NT-3 had minimal effects on dendritic growth (see alsoMcAllister et al. 1995). However, when slices were treated with both factors together, exogenous NT-3 blocked the growth-promoting effects of exogenous BDNF. Dendritic arbors of neurons treated with both factors were similar to those of untreated cells and distinct from those of BDNF-treated cells. Scale bar, 20 μm. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 8 NT-3, but Not NGF, Prevents Dendritic Growth Elicited by Exogenous BDNF Changes in dendritic complexity (DMI) for neurons from slices treated with either NT-3, BDNF, or NT-3 and BDNF together, at the indicated concentrations (A), and either NGF (200 ng/ml), BDNF (200 ng/ml), or NGF and BDNF together (B). (A) Even a 10-fold lower concentration of exogenous NT-3 (20 ng/ml) completely blocked the enhancement of dendritic complexity by 200 ng/ml of BDNF. (B) Exogenous NGF had no effect on the dendritic enhancement elicited by exogenous BDNF. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 9 Opposing Actions of BDNF and NT-3 in Regulating Dendritic Growth of Layer 6 Neurons Camera lucida reconstructions of basal dendritic arbors of neurons from untreated slices and slices treated with Trk–IgGs (20 μg/ml) and exogenous neurotrophins (200 ng/ml), as indicated. Neutralizing endogenous NT-3 with TrkC–IgG decreased dendritic growth, while neutralizing endogenous TrkB ligands with TrkB–IgG enhanced dendritic growth. TrkC–IgG completely prevented the dendritic growth elicited by TrkB–IgG when both receptor bodies were added together. Exogenous BDNF and NT-3 interacted similarly. Treatment with BDNF alone decreased dendritic elaboration, while treatment with NT-3 alone enhanced dendritic growth; NT-3- induced dendritic growth was completely prevented by exogenous BDNF. Scale bar, 30 μm. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 10 Opposing Effects of NT-3 and BDNF in Layer 6 Changes in dendritic complexity (DMI) for neurons from slices treated with Trk–IgGs and exogenous neurotrophins, as indicated. (A) Increases in dendritic complexity elicited by neutralizing endogenous TrkB ligands with TrkB–IgG were completely prevented by blocking endogenous NT-3 with TrkC–IgG. Conversely, the decreases in dendritic complexity caused by TrkC–IgG were largely prevented by TrkB–IgG. (B) Exogenous BDNF completely prevented the large increases in dendritic complexity elicited by NT-3 (200 ng/ml), even at a 10-fold lower concentration of BDNF (20 ng/ml). Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)

Figure 11 BDNF and NT-3 Act Directly on Layer 4 Neurons Two hours after acute dissociation, layer 4 neurons were treated with either 60 mM KCl + 10 μM forskolin for 5 min, or 200 ng/ml BDNF, 200 ng/ml NT-3, or combinations of the neurotrophins as indicated for 30 min. Neurons were immediately fixed and stained with an antibody against phosphorylated CREB to determine their ability to respond to neurotrophins. (A) Representative dissociated layer 4 neurons from each of the treatments. KCl + forskolin, BDNF, and NT-3 all caused phosphorylation of CREB, as indicated by the darkly stained nuclei in these photomicrographs. Thus, layer 4 neurons respond directly to both BDNF and NT-3. (B) Percentage of neurons staining positive for phospho-CREB following each treatment. Both BDNF and NT-3 induced CREB phosphorylation in about half of the cells, while few untreated cells stained positive for phospho-CREB. Treatment with both BDNF and NT-3 did not increase numbers of phospho-CREB positive cells over those induced by either factor alone, indicating that BDNF and NT-3 act on largely overlapping, if not identical, populations of neurons in layer 4. Neuron 1997 18, 767-778DOI: (10.1016/S0896-6273(00)80316-5)