a. Section from pleural ganglion (20X) showing the sensory neuron cluster. Sensory neurons (SNs) are mostly within the circle on the left side of the image (see small nuclei stained with DAPI). Other pleural neurons different than sensory neurons can be observed (larger nuclei on the right side of the image). ApGT1 staining (green) is observed at high intensity around sensory neurons and other neurons as well. The pleural-pedal connective (arrowheads) can be visualized as fibers stained with ApGT1 antibody. Small nuclei corresponding to glial cells (white arrows) in connectives as well as surrounding neurons are immunostained. In contrast, small nuclei (yellow arrows) found in the sheath of the ganglion (surrounding the ganglia) do not stain. b. Section of a pedal ganglion (20X) showing a few pedal neurons (PN, large DAPI-stained nuclei) as well as a portion of the neuropil (bottom left corner). Pedal neurons show no cytoplasmic staining for ApGT1 but ApGT1 staining surrounds them, presumably associated with glial cells (white arrows, see small nuclei surrounding neurons). Pedal neuropil also stained for ApGT1 (see bottom left corner), presumably associated with glial cells and neuronal terminals. c. A portion of a section of the sensory neuron cluster taken at a high magnification (60x) showing sensory neuron cell bodies (SN) as well as their axons and processes (arrowheads point to an axon coming from cell with *). ApGT1 immunostaining is associated with sensory neurons as well as glial cells (white arrows, see smaller nuclei). d. Section of pedal ganglia at high magnification showing pedal neurons (N1, N2). Section is cut through the middle of N1 (DAPI-stained nucleus) but through the periphery of N2 (notice the lack of DAPI staining). ApGT1 staining is not associated with those neurons (N1 cytoplasm is not stained with ApGT1) but rather ApGT1 antibody stained glial cells (white arrows) and indentations of glial processes into the neuronal cell bodies (red arrows, see ApGT1-stained processes). Scale bars are 50 μm in a,b and 10 μm in c, d. e. Section of pedal ganglia at low magnification stained without antibody but with DAPI and secondary antibody anti-rabbit-IgG Alexa-488 only. f. Pleural tissues contain much more ApGT1 per protein than pedal tissues. A western blot for ApGT1 shows the relative amounts of ApGT1 in sensory neuron clusters (SN cluster), the remaining pleural (Re Pleural), the pedal neuropil (Pe neuropil) and the remaining pedal (Re Pedal).

a. Example of an autoradiograph and Western blot of an IP from isolated pleural ganglia and isolated pedal ganglia incubated for 12 h in label (left side). Note that protein bands at the top of the western blots appear to correspond to ApGT1 multimers. Ab indicates the band corresponding to the antibody used in the IP that is detected by the secondary antibody in Western blots. Summary data (n = 7, right side). The values of labeled ApGT1 were normalized to total ApGT1 and were made relative to the lowest values (values of Isolated Pedal). ApGT1 was synthesized at higher rates in pleural than in pedal ganglia. b. An example of an autoradiograph and Western blot of an IP for SN clusters and the Re Pleural ganglia incubated for 12 h in label (left side). Note that about the same amount of labeled ApGT1 is observed in SN clusters and Re pleural but much more ApGT1 is found in Re Pleural because the amount of total protein obtained is greater than in SN clusters. Quantification of the results of the experiment in b (right side). ApGT1 was synthesized at higher rates in SN clusters than in the remainder of pleural ganglia.

a. Diagram of the experimental protocol. Pleural-pedal ganglia were removed, starved for methionine and cysteine for 3 h and then incubated in label overall for 8 h. In experimental groups, 5-HT (50 μM) was added for 2 h. After labeling, ganglia were incubated in culture media for 12-15 h to allow trafficking of newly synthesized ApGT1 to the terminals. b. Results from one experiment. IP was started with the same amount of protein in all groups (15 - 25 μg). c. Summary of quantification. The level of labeled ApGT1 in synaptosomes was not affected by 5-HT. However, in aliquots taken from the same samples, total synaptosomal ApGT1 before and after IP was significantly increased by 5-HT (n = 4). Therefore, the 5-HT induced increase in ApGT1 in pedal synaptosomes does not appear to be due to an increase in synthesis of ApGT1. d. To investigate whether 5-HT induced changes in pleural ganglia, levels of newly synthesized ApGT1 were measured in IP’s from pleural membranes 20 h after treatment of pleural- pedal ganglia with 5-HT. An example of a ³⁵S autorad of an IP sample and a summary of quantification of experiments are shown. Newly synthesized ApGT1 was significantly decreased in pleural ganglia 20 h after 5-HT.

Isolated pleural-pedal ganglia were treated in vitro with 5-HT and an inhibitor of vesicular transport Brefeldin A (18 μM) was applied immediately after the 5-HT treatment. Levels of ApGT1 were measured 24 h after treatments by western blotting. Brefeldin A alone did not have an effect on levels of ApGT1 (n = 7). ApGT1 was increased when ganglia were treated with 5-HT (n = 4). Brefeldin A added after 5-HT treatment (n = 5) blocked the increase in ApGT1 by 5-HT.

a. A section showing a sensory neuron with its axon and collaterals stained for ApGT1 and DAPI. ApGT1 staining can be observed in the sensory neuron (SN) cell body and varicosities (see arrowheads). Some small glial cells stained with ApGT1 are found attached to the cell body and processes (see arrows). b1. Image of three sensory neurons in culture taken at higher magnification. One confocal section through the middle of the cells showing ApGT1 immunostaining in the periphery of sensory neuron cell bodies. ApGT1 immunostaining was punctate and was associated with the cytoplasm but not the nucleus of sensory neurons. b2. Maximal projection image of ApGT1 immunostaining in same sensory neurons. c. Maximal projection image of a sensory cell showing no staining after peptide competition with the anti-ApGT1 antibody.

Pleural-pedal ganglia were incubated in label for 12 h, then protein synthesis was inhibited by addition of anisomycin (20 μM) and ganglia were left in culture for different chase periods of 0 h, 6 h and 12 h (attached pleural-pedal ganglia). Synaptosomes were then prepared from pedal ganglia and proteins were immunoprecipitated with ApGT1 antibody. a. Diagram of the experimental protocol and a representative example of an autoradiograph and Western blot. ACOP (ApGT1 co-precipitating protein) refers to a band running lower than ApGT1. Protein bands at the top of the western blots appear to correspond to ApGT1 multimers. b. Summary of quantification of all experiments (0h: n = 10, 6h: n = 5, 12h: n = 8). Values were relative to 0 h (basal value when the chase period began, indicated by the dashed line). Labeled ApGT1 was observed in synaptosomes by 12 h of labeling and 0 h of chasing and levels increased in a protein synthesis-independent manner after chasing for 6 and 12 h, suggesting that ApGT1 was transported to terminals. c. Colchicine (10 mM) was added to one group during the 12 h chase period. Also, pleural ganglia were removed from the pedal ganglia in one group after 12 h of labeling and the isolated pedal ganglia were then chased for 12 h. Values were relative to 0 h (dashed line). Addition of colchicine completely abolished the increase of labeled ApGT1 after chasing for 12 h, suggesting that ApGT1 was transported to the terminals via microtubule-dependent axonal transport. Removal of pleural ganglia significantly reduced the transported ApGT1 by about 170%. Thus, pleural ganglia (presumably sensory neuron cell bodies) appear to be a major source of synaptosomal ApGT1 in pedal ganglia.

a. (Left) Glutamate uptake was increased significantly by 27 % in pedal synaptosomes prepared 24h after isolated pedal ganglia were treated with 5-HT (50 μM/1.5 h). DRB, an inhibitor of transcription, added 30 minutes before 5-HT to the end of the experiment did not block the long-term increase in glutamate uptake produced by 5-HT (n = 9). DRB alone had no significant effect on basal glutamate uptake (n = 7). ANOVA followed by Newman Keul’s post hoc test (F(2,28) = 4.3, 5-HT vs 5-HT + DRB, p > 0.05; 5-HT vs DRB, p < 0.01; DRB vs 5-HT + DRB, p < 0.05). Asterisk (*) indicates significant % change (p < 0.05) from the control. (Right) Attached pleural-pedal ganglia were treated with 5-HT (50 μM) for 1.5 h. Pedal ganglia were separated from pleural ganglia 24 h after treatment and glutamate uptake was measured in synaptosomes derived from pedal ganglia. 5-HT significantly increased glutamate uptake by 45 % (n = 6, t = 5.7, p < 0.01, paired t-test). b. 5-HT did not produce long-term increases in glutamate uptake in cultures of isolated processes of sensory neurons. Sensory neuron cell bodies were excised and sensory neuron processes were treated with 5-HT (50 μM) for 1.5 h. Glutamate uptake measured 24 h after treatment of sensory neuron processes with 5-HT did not change. In contrast, cultures of intact isolated sensory neurons treated with 50 μM for 1.5 h produced significant long-term increases in glutamate uptake.

Isolated pleural-pedal ganglia were treated with 5-HT for 2 h and 24 h later pleural-pedal membranes were prepared, run in a 2D gel, transferred to PVDF and subsequently blotted for ApGT1. a. Western blots with spots of proteins corresponding to ApGT1 are shown in control samples and 5-HT treated samples. Extensive heterogeneity of charge is observed for ApGT1 as well as a small heterogeneity of molecular weight. Comparison of ApGT1 from control and 5-HT samples revealed that some spots of ApGT1 found in the control disappeared or decreased after 5-HT treatment (red boxes; spots # 8, 9, 10, 11) and some new spots of ApGT1 appeared or increased after 5-HT treatment (yellow boxes; spots # 1, 2, 3, 4, 5, 6, 7). b. Quantification of experiment in a. Overall, it appears that a shift of the isoforms for ApGT1 towards acidic pH occurred suggesting that 5-HT produced a change in post-translational modifications of ApGT1.