Figure 3. The Rapid Increase in Sensorin Expression Induced by 5-HT Is Blocked by Postsynaptic BAPTA.
(A) Phase contrast micrograph of a sensorimotor coculture. The arrow points to the major neurite of the motor neuron. Scale bar, 30µm.
(B) Micrographs of sensorin immunofluorescence in the experimental groups. Each micrograph depicts the region along the main axon of a sensory neuron where contacted the major neurite of the motor neuron. Previous evidence indicates that this region represents the area of maximum synaptic contact between the sensory and motor neurons [40]. In cocultures that did not receive a postsynaptic injection of BAPTA, 5-HT treatment increased sensorin staining compared to untreated Controls. In cocultures treated with 5-HT after a postsynaptic BAPTA injection the immunostaining resembled that in Controls. Scale bar, 10 µM.
(C) Intensity of sensorin immunostaining in the four experimental groups. Staining intensity was determined for each coculture by measuring the mean pixel intensity of the fluorescence in four circular regions centered on the main process of the motor neuron. The mean pixel intensity was then corrected for background fluorescence, and the result was normalized to the mean pixel intensity in Control group (Figure S1). A one-way ANOVA indicated that the group differences were highly significant [F(3,82) = 6.13, p = 0.0008]. A Post-hoc comparison showed that the normalized sensorin staining was greater in 5-HT cocultures (151 ± 15%, n = 20) than in Controls (100 ± 5%, n = 26; p < 0.01). Importantly, the mean normalized sensorin staining in 5-HT-BAPTA cocultures (97 ± 6%, n = 20) was significantly less than that in the 5-HT group (p < 0.01), and not statistically different from that in Control cocultures (p > 0.05). The postsynaptic BAPTA injection alone had little effect on sensorin staining (mean normalized staining in the BAPTA group = 111 ± 8%, n = 20; p > 0.05 for the comparison with the Controls). *, significance of the difference between 5-HT and Control groups; +, significance of the difference 5-HT and 5-HT-BAPTA groups. Error bars represent ± SEM.