Retrograde repression of growth-associated protein-43 mRNA expression in rat cortical neurons

Abstract

Corticospinal neurons support rapid growth of axons toward spinal cord targets in the perinatal period. Initial axon growth is accompanied by elevated expression of growth-associated protein-43 (GAP-43), which then declines in postnatal development. To investigate whether expression of GAP-43 mRNA is regulated by retrograde signals, we injected colchicine into the corticospinal tract to block retrograde axonal transport during a time when GAP-43 is normally declining in corticospinal neurons. Colchicine caused a prolongation of high GAP-43 mRNA expression in neurons located in layer V (but not other layers) of sensorimotor cortex. We next used osmotic minipumps to infuse soluble adult spinal cord extract into the sensorimotor cortex. This resulted in a premature downregulation of GAP-43 mRNA in identified corticospinal neurons. GAP-43 repressive activity was found in extracts of the spinal cord tissue as young as postnatal day 8. The effect of spinal cord extract in vivo was not mimicked by adult cerebellar or muscle extracts. Cultures of postnatal cortical neurons also underwent downregulation of GAP-43 mRNA when treated with spinal cord extract. Activation of cAMP signaling also repressed GAP-43 mRNA in cortical cultures, and the repressive effect of spinal cord extract was diminished by an adenyl cyclase inhibitor. Thus, GAP-43 mRNA may be downregulated late in development by a target-derived retrograde repressive factor, and this effect may be mediated by cAMP second messenger signaling.

Fig. 1.

Blocking axonal transport prevents developmental downregulation of GAP-43 in layer V cortical neurons. A,Sensorimotor region of cerebral cortex at P28 stained with cresyl violet and showing cortical layers. Scale bar, 150 μm. B, C,In situ hybridization for GAP-43 in layer V neurons at P28. Animal was injected with vehicle alone (B) or colchicine (C) at P23. Scale bar (in C), 25 μm. D,Quantification of GAP-43 mRNA in situ hybridization signal in neurons of different cortical layers on P28. Error bars indicate SEM. Colchicine injection resulted in significantly higher expression of GAP-43 mRNA in layer V neurons (*p < 0.0001; Student's t test), but not in other layers (p > 0.05; Student's ttest).

Fig. 2.

Infusion of extract from adult spinal cord represses GAP-43 mRNA in CST neurons. A, B,Electronically combined images of FB-labeled CST neurons and silver grains from in situ hybridization after chronic infusion of saline (A) or adult spinal cord extract (B). Scale bar (in B), 50 μm. Quantitative in situ hybridization signal for GAP-43 mRNA in CST neurons identified by FB retrograde labeling at P18 (C) showed that there was a significant reduction of GAP-43 mRNA after infusion of adult spinal cord extract compared with infusion of saline or saline plus BSA (*p < 0.001; one-way ANOVA followed by Tukey's test). Error bars indicate SEM.

Fig. 3.

Infusion of extract from immature spinal cord represses GAP-43 mRNA in CST neurons. Quantitative in situ hybridization for GAP-43 in CST neurons identified by Fast Blue retrograde labeling at P18 showed that chronic infusion of soluble extracts from adult, P8, P14, and P21 spinal cord tissue caused similar significant reductions in GAP-43 mRNA compared with infusion of saline plus BSA (*p < 0.001; one-way ANOVA followed by Tukey's test). There was no significant difference between the effects of adult, P8, P14, and P21 spinal cord extracts (p > 0.05; ANOVA). Infusion of extracts from adult cerebellum or skeletal muscle caused no change in GAP-43 mRNA levels in CST neurons (p > 0.05; ANOVA). Error bars indicate SEM.

Fig. 4.

Extract from adult or immature spinal cord represses GAP-43 mRNA in cultured cerebral cortical neurons. Quantitative in situ hybridization for GAP-43 in cultures prepared from P8 cortex revealed that exposure to extracts from adult, P8, P14, and P21 spinal cord tissue caused significant reductions in GAP-43 mRNA compared with control after 5 d in culture (*p < 0.001; one-way ANOVA followed by Tukey's test). The effect caused by extract from adult cerebellum was significantly different from both control and adult spinal cord extract (*p < 0.001 for both comparisons; Tukey's test). There was no significant difference among the effects of adult and postnatal spinal cord extracts on GAP-43 expression (p > 0.05; one-way ANOVA). Error bars indicate SEM.

Fig. 5.

Activation of the cAMP signaling represses GAP-43 expression in cultured cerebral cortical neurons. Quantitativein situ hybridization for GAP-43 in cultures prepared from P8 cortex showed that exposure to either adult spinal cord extract or 0.5 mm dBcAMP caused a significant reduction in GAP-43 mRNA compared with control cultures after 6 d in culture (*p < 0.001; one-way ANOVA followed by Tukey's test). Exposure to 0.2 mm adenyl cyclase inhibitor SQ22,536 alone caused a small apparent reduction in GAP-43 expression, but this was not significant (p > 0.05; Tukey's test). Cultures treated with both spinal cord extract and SQ22,536 showed a significantly greater level of GAP-43 expression than cultures treated with spinal cord extract alone (**p < 0.001; Tukey's test). Error bars indicate SEM.