A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat

Abstract

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter-IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Figure 1

Comparative effects of C(92-96) and mAb NGF30 on NGF-increased cholinergic activity of septal neurons. NGF (1 ng/ml), C(92-96) (100 μg/ml), and/or mAb NGF 30 (10 ng/ml) were added to the culture medium 1 day after plating. ChAT activity was measured on the fourth, sixth and eighth days after plating (4, 6, and 8 DIV). Results are means ± SEM of four different experiments, ∗, P < 0.05 vs. NGF alone; ∗∗, P < 0.01 vs. NGF alone.

Figure 2

Synaptophysin (A, B, and C) and VAChT (D, E, and F) immunoreactivity in the ipsilateral hindlimb cortex of control peptide (A and D)-, mAb NGF30 (B and E)-, and C(92-96) (C and F)-infused animals. Note the apparent reduction in the number of VAChT-IR in E and F compared with D. (Bar = 20 μm.)

Figure 3

Effect of cortical injections of C(92-96) and mAb NGF30 on the number of synaptophysin-IR sites in the hindlimb and in the parietal I cortex. The drugs were injected into the hindlimb cortex as indicated in Materials and Methods. Twelve 8,500-μm² sections were digitized from five consecutive sections in each cortical area of each rat (five animals per group). The number of synaptophysin-IR sites in 1,000 μm² is expressed as the mean ± SEM. HL, hindlimb; Par I, parietal I.

Figure 4

Effect of cortical injection of C(92-96) and mAb NGF30 on the number (A) and on the size (B) of VAChT-IR sites in the hindlimb and in the parietal I and II cortices. Note in A the loss of cholinergic boutons (VAChT-IR), which is more prominent with the TrkA antagonist C(92-96) and is related to the distance from the injection site; B illustrates the shrinkage of the cholinergic boutons after the administration of both compounds. The drugs were injected into the hindlimb cortex as indicated in Materials and Methods. Twelve 8,500-μm² sections were digitized from five consecutive sections in each cortical area of each rat (five animals per group). The number of VAChT-IR sites (A) in 1,000 μm² is expressed as the mean ± SEM. The size of VAChT-IR sites (B) (in square micrometers) is expressed as the mean ± SEM. HL, hindlimb; Par II, parietal II. ∗, P < 0.05 vs. each control peptide area; ∗∗, P < 0.01 vs. each control peptide area.

Figure 5

Cortical injections of C(92-96) and mAb NGF30 did not affect the size of VAChT positive neurons of the nucleus basalis. The drugs were injected into the hindlimb cortex as indicated in Materials and Methods. Six sections from the midbasalis were taken from each animal (five animals in each group). Three random fields from each level were quantified. The size of VAChT neurons (in square micrometers) is expressed as the mean ± SEM.