A novel role for p75NTR in subplate growth cone complexity and visual thalamocortical innervation

Abstract

In cortical development, subplate axons pioneer the pathway from neocortex to the internal capsule, leading to the proposal that they are required for subsequent area-specific innervation of cortex by thalamic axons. A role for p75 neutrophin receptor (NTR) in area-specific thalamic innervation of cortex is suggested by the observation that p75NTR expression is restricted to subplate neurons in a low-rostral to high-caudal gradient throughout the period of thalamocortical innervation. In vitro, neurotrophin 3 binding to p75NTR increases neurite length and filopodial formation of immunopurified subplate neurons, suggesting a role for p75NTR in subplate growth cone morphology and function in vivo. Consistent with this idea, subplate growth cones have markedly fewer filopodia in mice lacking p75NTR than in wild type mice. Despite this gross morphologic defect, many subplate axons in knock-out mice pioneer the projection to the internal capsule as they do in wild-type mice. However a few subplate axons in the knock-out mice make ectopic projections rostral in the intermediate zone and frontal cortex. Concomitant with the altered morphology of subplate growth cones, mice lacking p75NTR have diminished innervation of visual cortex from the lateral geniculate nucleus, with markedly reduced or absent connections in 48% of knock-out mice. Thalamic projections to auditory and somatosensory cortex are normal, consistent with the gradient of p75NTR expression. Our present results are unusual in that they argue that p75NTR functions in a novel way in subplate neurons, that is, in growth cone morphology and function rather than in axon extension or neuronal survival.

Fig. 1.

p75NTR expression in subplate during development. An in situ probe specific for exon III of p75NTR was used to survey p75NTR expression. High-magnification view of subplate p75NTR expression in cross section of cortex at ages from E14.5 through P14. PP, Preplate; VZ, ventricular zone;SP, subplate; m, meninges;MZ, marginal zone; SVZ, subventricular zone; CCP, condensed cortical plate; WM, white matter.

Fig. 2.

Blockade of NT3 binding to p75NTR reduces neurite length and filopodial number in cultured subplate neurons. Immunopurified subplate neurons were cultured on fibronectin in serum-free media with addition of BDNF (▴), NT3 (▪), NT3 + anti-p75NTR FAb (■), or control (no neurotrophin) (●).A, After 5 d in vitro, the length of the longest neurite was measured and plotted against percentage of cells with length > X. B, After 2 d in vitro, the total number of filopodia per neuron was measured and plotted versus percentage of neurons with > X total filopodia.

Fig. 3.

Abnormal subplate neuron growth cones in p75NTR knock-out mice. Subplate neuron growth cones were labeled at E13.5 with crystals of DiI inserted into developing visual cortex.A, Individual growth cones are imaged in both genotypes in the internal capsule, at the leading edge of labeled subplate projection (boxed area). B, Filopodia per growth cone measured for wild-type (●) and knock-out (○) mice. C, D, Representative examples of growth cone morphology from wild-type (C) or knock-out mice (D) are marked witharrows.

Fig. 4.

Subplate axons make pathfinding errors in p75NTR knock-out mice. Crystals of DiI were placed in presumptive visual cortex at E15.5, before LGN axons have arrived underneath caudal cortex, to label subplate neurons selectively. A,B, Coronal sections show DiI-labeled subplate axons reaching the internal capsule (IC) in wild-type mice (A) or p75NTR knock-out mice (B). C, D, In slightly more rostral sections, ectopic subplate axons were found in the p75NTR knock-out mice (D) but not wild-type mice (C), some of which turn lateral (arrowheads in boxed area) to enter the preplate (PP). GE, Ganglionic eminence;IZ, intermediate zone; AC, anterior commissure.

Fig. 5.

Gradient of p75NTR expression in subplate during development. A–D, Mid-sagittal sections at E14.5 (A), E16.5 (B), P1 (C), and P7 (D) demonstrate a low-rostral high-caudal gradient of p75NTR expression.Arrowheads denote rostral–caudal extent of p75NTR expression. Other structures with p75NTR expression are labeled or marked with arrow or asterisk.PT, Posterior thalamus; RT, reticular thalamus; cb, cerebellum; BFC, basal forebrain complex.

Fig. 6.

Reduced visual thalamocortical innervation in p75NTR knock-out mice. A, B, DiI (red) and DiD (green) crystal placement in auditory cortex and visual cortex, respectively, in wild-type (A) and knock-out (B) mice at P10. C,D, Retrograde labeling of cells and anterograde labeling of fibers in LGN and thalamus after cortical labeling shown inA and B in coronal sections. DiD (green) from visual cortex heavily labels the LGN (outlined in white) in wild-type (C) but not knock-out (D) mice. DiI (red) from auditory cortex labels fibers of passage heading to the MGN (shown in Fig. 8), as well as some cells of the ventrobasal nucleus of thalamus. E,F, Anterograde labeling of LGN axons and retrograde labeling of cortical cells after DiI (red) placement in the lateral dorsal thalamus. In these sagittal sections (caudal is to the left), labeled thalamic axons project from the LGN to the internal capsule (IC), then within the intermediate zone (IZ) toward visual cortex (denoted between arrowheads) in wild-type (E) but not knock-out (F) mice.

Fig. 7.

Transneuronal transport confirms diminished geniculocortical projection in p75NTR knock-out mice.A, B, Dark-field autoradiography showing transneuronal transport of ³H-proline to LGN terminals in visual cortex of adult (>P90) mice in sagittal sections (caudal is to the left). Arrows denote visual cortex in wild-type (A) and knock-out (B) mice. C, Quantification of innervation. Data points show innervation index (see Materials and Methods) in all wild-type and knock-out mice examined.Arrows indicate mice used in A andB.

Fig. 8.

Normal auditory and somatosensory thalamocortical innervation. A, B, Retrograde labeling of cells in MGN (outlined in white) after DiI placement in auditory cortex (for location of DiI crystal placement, see Fig. 6A,B) in wild-type (A) and knock-out (B) mice at P10. Sections are coronal.C, D, Serotonin immunohistochemistry in coronal sections at the level of somatosensory cortex of wild-type (C) and knock-out (D) mice. Patches of staining indicated by arrowheads show thalamic axon terminals in the barrel representations of whisker vibrissae.

Fig. 9.

Subplate neuron generation and cell death are similar in p75NTR knock-out and wild-type mice.A–I, Coronal sections of P2 wild-type (A–C) and p75NTR knock-out mice (D–I) were double labeled with BrdU immunohistochemistry (green) and ISEL (red). A, D, Low-magnification views of BrdU immunohistochemistry performed in P2 wild-type (A) and knock-out mice (D) after injection of BrdU at E12.5 to label subplate neurons at their birth. B, E, ISEL staining of the same sections in wild-type (B) as compared with knock-out mice (E). Examples of dying cells in subplate (asterisk in B; arrow inE) can be seen in both genotypes. C,F, Overlay of BrdU labeling and ISEL confirms that subplate neurons undergo DNA fragmentation, consistent with programmed cell death (blue label is bisbenzamide nuclear counterstain). G–I, High-magnification image of a double-labeled cell (asterisk inG–I) along with single-labeled BrdU-positive (horizontal arrow in G,I) and ISEL-positive (vertical arrow in H, I) cell.

Fig. 10.

Developmental changes in subplate neuron cell counts and ISEL are similar in p75NTR knock-out and wild-type mice. Subplate neurons labeled at E12.5 with BrdU were visualized at E18.5, P1, P3, P7, P14, and P21 with BrdU immunohistochemistry (see Fig. 9). Dying cells with DNA strand breaks are detected with ISEL in adjacent sections. Mean cell counts from three coronal levels were binned at E18.5/P1 (onset of subplate neuron cell death) and P3/P7 (peak of cell death) time points (see Materials and Methods). Mean cells per section is plotted ±SD. BrdU-immunopositive subplate neuron number and ISEL-positive cell death are not significantly different between knock-out and wild-type mice at any age.