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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: J Neurosci. 2010 Jun 23;30(25):8353–8366. doi: 10.1523/JNEUROSCI.0546-10.2010

Figure 3.

Figure 3

Down regulating ApCAM from the surfaces of L7 blocks initiation of synapse formation, L7-induced regulation of sensorin expression in the cell body of the sensory neurons and synapse-associated growth. A. Phase contrast and epifluorescent images of sensorin immunostaining in sensory neurons after 6 h of contact with L7 (n = 10 for control and n = 12 for 4E8) or L11 (n = 8 for each treatment) that were plated the previous day and pre-incubated with control or 4E8 antibody. Antibodies were washed out and sensory neurons plated about 1 h later. Sensorin staining in the sensory neuron cell body was low following treatment of L7 with 4E8. Also note that there was no growth from the axon stump of the sensory neuron. Treatment of L11 with 4E8 did not interfere with sensorin expression in the cell body, axon or neurites of the sensory neurons. The bar equals 50 μm. B. Down regulating ApCAM on L7 with 4E8 blocked synapse formation at 6 h, and significantly reduced synapse strength when EPSP were recorded again at 20 h. The height of each bar is the Mean ± SEM of the EPSP amplitude measured at 6 h and 20 h for control- and 4E8-treatment of L7 prior to the addition of the sensory neuron. ANOVA indicated a significant effect of treatment (df = 1, 26; F = 7.979, p < 0.01). Individual comparisons indicated a significant reduction in EPSP with treatment with 4E8 at both 6 h and 20 h (p < 0.01). C. Down regulating ApCAM on L7 with 4E8 blocked the increase in sensorin expression, especially in the cell body, while treatment of L11 with 4E8 did not interfere with sensorin expression throughout the sensory neuron. The height of each bar is the Mean ± SEM staining intensity in the various compartments of the sensory cell normalized to the staining intensity in each compartment for control L7 + SN cultures (100%). Since no varicosities formed with pretreatment of L7 with 4E8, there is no bar for that compartment. ANOVA indicated a significant effect of treatment (df = 6, 72; F = 195.526, p < 0.001). Individual comparisons indicated that treatment with 4E8 resulted in a significant reduction in sensorin expression in the cell body of sensory neurons contacting L7 (p < 0.01), but had no effect on expression in sensory neuron axons. There was no significant difference in the treatment of L11 with 4E8; both 4E8 and control treatments did not alter the significant reduction (p < 0.05) in sensorin staining in the cell body and original axons. D. Down regulating ApCAM on the surface of L7 significantly blocked neuritic growth by sensory neurons, but down regulating ApCAM on the surface of L11 did not affect neuritic growth by sensory neurons. The height of each bar is the Mean ± SEM in the extent of neuritic growth of sensory neurons in contact with L7 or L11 based on detectable sensorin immunochemistry in the distal neurites of the sensory neurons. Treatment of L7 with 4E8 blocked neuritic growth by sensory neurons. This was also verified by injection with carboxyfluoroscein (n = 3, data not shown). ANOVA indicated a significant effect of treatment (df = 3, 36; F = 62.015, p < 0.001). Individual comparisons indicated that pretreatment of L7 with 4E8 significantly reduced growth (p < 0.01) while pretreatment of L11 with 4E8 did not affect growth by sensory neurons contacting L11.