An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact

Abstract

Neurotrophin-3 (NT-3), its cognate receptor trkC, and voltage-gated calcium channels are coexpressed by embryonic pyramidal neurons before target contact, but their functions at this stage of development are still unclear. We show here that, in vitro, anti-NT-3 and anti-trkC antibodies blocked the increase, and NT-3 reversed the decrease in the number of calbindin-D(28k)-positive pyramidal neurons induced by, respectively, calcium channel activations and blockades. Similar results were obtained with single-neuron microcultures. In addition, voltage-gated calcium channel inhibition downregulates the extracellular levels of NT-3 in high-density cultures. Moreover, electrophysiological experiments in single-cell cultures reveal a tetrodotoxin-sensitive spontaneous electrical activity allowing voltage-gated calcium channel activation. The mouse NT-3 (-/-) mutation decreases by 40% the number of developing calbindin-D(28k)-positive pyramidal neurons, without affecting neuronal survival, both in vitro and in vivo. Thus, present results strongly support that an activity-dependent autocrine NT-3 loop provides a local, intrinsic mechanism by which, before target contact, hippocampal pyramidal-like neurons may regulate their own differentiation, a role that may be important during early CNS differentiation or after adult target disruption.

Fig. 1.

Endogenous NT-3 promotes the expression of calbindin-D_28k of hippocampal pyramidal-like neurons after 6 d in vitro. Anti-NT-3- and anti-trkC antisera inhibit the constitutive increase in the number of calbindin-D_28k-positive neurons (A) without affecting neuronal survival (B). Number of calbindin-D_28k-positive neurons (C) and of total number of cells (D) from hippocampal dissociated cultures from E16 mice wild-type and NT-3 (−/−) mutant embryos in the absence (white) or presence (black) of 1 ng/ml NT-3 after 6 d in vitro. *p < 0.01.

Fig. 2.

Endogenous NT-3 promotes the expression of calbindin-D_28k of hippocampal pyramidal-like neuronsin vivo. A, B, Photomicrographs of equivalent cross sections of hippocampus from P0 wild-type (A) and NT-3 (−/−) mutant (B) mice stained with calbindin-D_28kantisera. py, Stratum pyramidale. The staining with calbindin-D_28k antibodies was lower, and pyramidal neurons appeared smaller in NT-3 (−/−) than in NT-3 (+/+) mice. Scale bar, 50 μm. C, D, Quantitative analysis of the number of calbindin-D_28k-positive neurons (C) and the total number of neurons (D) in the hippocampus derived from P0 wild-type and NT-3 (−/−) mutant mice. *p < 0.01.

Fig. 3.

Endogenous NT-3 mediates the effects of calcium channel activation on the expression of calbindin-D_28k of immature hippocampal pyramidal-like neurons in vitro. Addition of anti-trkC antibodies (0.4 μg/ml) to the 50 mmKCl-depolarizing medium inhibited the increase in the number of calbindin-D_28k-positive neurons induced by 1 hr daily stimulation during 5 d with 50 mm KCl (A). Chronic treatment for 5 d (1–6 DIV) with 10 ng/ml NT-3 induced an increase in calbindin-D_28k-positive neurons. In the presence of 500 nm nitrendipine (Nitr) or 250 nmω-agatoxin-IVA, NT-3 still induced increase in calbindin-D_28k-positive neurons (B). Chronic treatment for 5 d with 500 nm nitrendipine, 0.4 μg/ml anti-trkC alone, or both shows a similar 40% decrease in the number of calbindin-D_28k-positive pyramidal neurons compared with control conditions (C).

Fig. 4.

L- and Q-type, but not N-type, calcium channels expressed by immature hippocampal neurons regulate the extracellular levels of NT-3. Control measurements of the culture medium without cells showed no contamination with NT-3 (Neurobasal). ω-Agatoxin-IVA at 250 nm plus 500 nm nitrendipine during 3 DIV significantly reduced the extracellular levels of NT-3, whereas 1 μm ω-conotoxin-GVIA in the same condition had no effect compared with control conditions.

Fig. 5.

A calcium-dependent NT-3 autocrine loop promotes the expression of calbindin-D_28k of hippocampal pyramidal-like neurons. A, Bright-field micrograph of an embryonic hippocampal calbindin-D_28k-positive neuron growing in microwell culture as a single cell after 6 DIV in control conditions. Scale bar, 50 μm. Effect of anti-NT-3 and anti-trkC antisera and nitrendipine (500 nm; nitr) and ω-agatoxin-IVA (250 nm) on the percentage of calbindin-D_28k-positive neurons (B) and the percentage of pyramidal neurons survival (C) from hippocampal neuronal cultures as single cell (control, n = 100 wells; anti-trkC polyclonal antibody,n = 98 wells; anti-NT-3 polyclonal antibody,n = 195 wells; nitrendipine, n = 105 wells; ω-agatoxin-IVA, n = 110 wells from two separate experiments). *p < 0.01.

Fig. 6.

Single pyramidal-like neurons displayed a phasic spontaneous electrical activity. A, B, Bright-field micrograph of a single pyramidal-like neuron grown in very low-density culture recorded and stained at 5 DIV. Staining from the same neuron with neurofilament (A) and calbindin D_28k (B) antibodies. Scale bar, 25 μm. The corresponding electrical activity of the same neuron before staining recorded with the loose patch is shown in C as upward deflections. D, Spontaneous electrical activity recorded at 3 DIV on a pyramidal-like neuron grown as a single-neuron microculture. Application of 1 μm TTX inhibited electrical activity.