TrkB-like immunoreactivity is present on geniculocortical afferents in layer IV of kitten primary visual cortex

Abstract

Exogenous administration of the neurotrophins brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5), or blockade of their endogenous actions, have been reported to affect the anatomic organization and physiological responses of neurons in developing mammalian primary visual cortex. Experimental alteration of levels of these neurotrophic factors can also influence the morphology of the geniculocortical afferents that project from the lateral geniculate nucleus (LGN) to primary visual cortex. BDNF and NT-4/5 are ligands of the TrkB tyrosine kinase receptor. Although multiple populations of cortical neurons express TrkB, it is not known whether geniculocortical afferents express this receptor on their axon branches in visual cortex. We have anatomically labeled geniculocortical afferents of postnatal day 40 kittens with the anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin (PHA-L) and performed double-label immunofluorescence with a panel of anti-TrkB antibodies. Confocal microscopy and object-based colocalization analysis were used to measure levels of TrkB-like immunoreactivity (IR) on geniculocortical afferents in layer IV of primary visual cortex. By using a conservative analysis involving a comparison of measured colocalization with the amount of colocalization expected based on random overlap of TrkB puncta and PHA-L--labeled afferents, 3 of 5 anti-TrkB antibodies tested showed significant colocalization with the geniculocortical axons. Results for the other two antibodies were indeterminate. The indices obtained for colocalization of TrkB and geniculocortical afferents were also compared with the equivalent index obtained for GAD65, a protein that has a similar overall expression pattern to that of TrkB but is not expressed on geniculocortical axons. This analysis indicated that TrkB was present on geniculocortical axons for all five TrkB antibodies tested. TrkB-like IR was also observed on neuronal somata in the LGN. These results indicate that TrkB receptors on geniculocortical afferents are potential mediators of the actions of BDNF and NT-4/5 in developing visual cortex.

Fig. 1

Anti-TrkB antibodies shown on schematic TrkB receptor. The RTB antibody was raised against the biochemically purified extracellular domain of rat TrkB after heterologous expression in COS-7 cells and was used as an antiserum. The other anti-TrkB antibodies were generated by immunization with synthetic peptides corresponding to specific domains of the rat TrkB amino acid sequence and were affinity purified by using the same peptide. The TrkB606 antibody should recognize only the tyrosine kinase-containing full-length isoform, whereas the others should recognize both full-length and truncated isoforms.

Fig. 2

TrkB-like immunoreactivity (IR) is present on cell bodies in the lateral geniculate nucleus. False-color double-label immunofluorescent images are shown for TrkB23 (A), TrkB146 (B), TrkB348 (C), and TrkB606 (D). Red is TrkB-like IR, and green indicates MAP2-like IR, which strongly labels dendrites but also allows the visualization of the cytoplasmic region of cell bodies as diffuse label surrounding unlabeled cell nuclei. For all anti-TrkB antibodies, the label is primarily punctate in neuronal cell bodies and neuropil. Nuclear labeling is probably artifactual (see text). Scale bars = 10 μm in A-D.

Fig. 3

Visual image inspection is inadequate to determine whether TrkB-like immunoreactivity is colocalized with geniculocortical afferents. False-color double-label immunofluorescent confocal images from layer IV of kitten visual cortex are shown in red for TrkB23 (A), TrkB146 (B), TrkB348 (C), TrkB606 (D), RTB (E), and GAD65 (F) and in green for Phaseolus vulgaris leucoagglutinin (PHA-L)/-labeled geniculocortical afferent arbors (A-F). Although apparent colocalization of TrkB-positive puncta with PHA-L-labeled afferents can be observed for all antibodies, it is also present for GAD65. Scale bars = 1 μm in A-F.

Fig. 4

Quantitative colocalization analysis reveals the presence of TrkB-like immunoreactivity on geniculocortical afferents. The amount of colocalization of TrkB or GAD65 with the Phaseolus vulgaris leucoagglutinin-labeled afferents was computed by using either object- or pixel-based analyses and expressed as a contrast index (Silver and Stryker, 2000). Positive values of this index indicate colocalization, negative values indicate anticolocalization, and values not significantly different from 0 are indeterminate. A: Object-based analysis. B: Pixel-based analysis. Sample sizes are 18 pairs of images for TrkB606 and 12 pairs of images each for the other antibodies. Error bars represent standard errors of the difference, and P values based on one-tailed t tests are shown for each condition. Asterisks indicate statistical significance at the P < 0.05 level.