Table 1.
Agonists and antagonists used in calcium image and patch clamp experiments.
Figure 1.
Glutamate assay of dissociated DRG cultures.
Colorimetric assay shows that following KCl (100 µM) application to the culture medium there was an increase in glutamate release which did not reach significance compared to control (no stimulus/HEPES only). Following pretreatment with a glutamate transporter blocker (TBOA 100 µM), KCl stimulation resulted in a significant increase in extracellular glutamate concentration. Each column represents the mean of 3 wells per condition. Each column represents the mean ± S.E.M. One-way ANOVA **, P<0.01.
Figure 2.
Confocal and calcium influx images of a 24-hour DRG primary mixed culture.
A–C confocal image showing SGCs (red: glutamine synthetase an SGC marker, open arrow), neuron (green: β-tubulin, solid arrow) and nucleus (blue: DAPI). D–F representative pictures from a 24-hour culture used for calcium imaging. D and E (high magnification) were taken before the start of calcium imaging. F, fluorescent image following 200 µM glutamate application showing an increase in the 340/380 ratio indicated by an increase in signal intensity in the neuron (N), and two SGCs (yellow arrows) compared to the SGC which did not respond (white arrow). C, D, E, F, scale bar: 10 µm.
Figure 3.
Representative calcium imaging fluorescent traces of a neuron and SGC.
A, B, following application of 200 µM glutamate both neurons and SGCs showed an increased fluorescent ratio during the 10 minutes of recording. A1, B1 after washing with HEPES buffer, KCl (50 mM) was given to identify neurons in the recorded field. Ionomycin (20 µM) was added to test for cell viability at the end of the experiment. Lines show the duration of application for experimental agent. Scale bar = 5 minutes. C, average relative glutamate induced maximum change of 340/380 fluorescence ratio from pre-drug (baseline, normalized at 0) condition. Paired Student’s t- tests were used to compare pre-drug and post-drug conditions. Mean ± S.E.M. ***P<0.001.
Figure 4.
Representative calcium imaging traces of SGCs and neurons following application of glutamate agonists and antagonists.
Following direct application of A: AMPA (50 µM), B: NMDA (100 µM), C: DHPG (100 µM) and D: KA (30 µM) both neurons and SGCs responded to all three agonists. Following a HEPES buffer wash the same cells were then given the appropriate selective antagonist: A1, E1: CNQX (100 µM); B1, F1: APV (100 µM); C1, G1: AP3 (1 mM) and D1, H1: UBP310 (0.5 µM) for 5 minutes prior to second application of agonist. In the presence of its antagonist, the effect of each agonist was blocked (A1–D1 SGCs; E1–H1 neurons). A2–H2, ten minutes after HEPES buffer wash, KCl (50 mM) was given to identify neurons in the recorded field and ionomycin (20 µM) was added to test cell viability at the end of the experiment. Lines show the duration of application. Scale bar = 5 minutes.
Figure 5.
Summary data of calcium imaging experiment on glutamate receptor activation.
Effects of each agonist were significantly blocked in the presence of their respective antagonists (A, SGCs; B, neurons) in calcium imaging experiments. Pre-treatment of the selective appropriate antagonist, resulted in a reduction in activation by the agonist of both neurons and SGCs. Cell numbers used in this summary bar graph (neuron/SGC): AMPA (25/35), NMDA (12/9), DHPG (20/5) and KA (11/22). Each agonist used 3–4 cultures from 2–3 animals. Each column represents the mean ± S.E.M. ***, P<0.001; **P<0.01.
Table 2.
Neurons and SGCs responding to glutamate or selective agonists with or without specific antagonists using calcium imaging.
Figure 6.
Whole cell patch recordings of small neurons from ex-vivo dorsal root ganglia.
A–E, puff application of glutamate or the receptor selective agonists induced inward currents which were blocked (right trace) by bath application of the appropriate selective antagonist(s). A1–E1, bar graphs showing the reduction in standardized amplitude of the receptor induced currents in presence of the appropriate antagonist. See Table 1 for drug concentrations. The bars above all the traces indicate agonist application time (200 ms). The decay time for AMPA induced current is: 520.0±70.4 ms; NMDA: 461.9±39.7 ms; Glutamate: 537.0±50.8 ms; KA: 512.4±482.4 ms; DHPG: 90.1±25.8 ms. All data are expressed as mean ± SEM. ***P<0.001.
Table 3.
Neurons responding to glutamate or selective receptor agonists in ex-vivo patch-clamp experiments.
Table 4.
Neurons responding to selective antagonist following agonist treatment.
Figure 7.
Representative images from an L4 DRG immunostained for glutamate following CCI of the sciatic nerve.
Increased glutamate immunolabeling is seen in the DRG ipsilateral to the sciatic CCI (A standard, C thresholded image) compared to the contralateral side (B standard, and D thresholded image). E, F shows the % difference in glutamate immuno-expression between the ipsi and contralateral L3 - L6 DRGs at 7 (E) and 21 (F) day post-CCI-SN animals. Scale bar = 50 µm. Data expressed as mean ± SEM. **, P<0.01; ***P<0.001 ipsilateral vs. contralateral.
Figure 8.
Representative images from a trigeminal ganglion immunostained for glutamate following CCI of the ION.
Increased glutamate immunolabeling is seen in the trigeminal ganglion ipsilateral to the CCI (A, standard, C, thresholded image) compared to the contralateral side (B, standard, D, thresholded image). I, shows the % difference in glutamate immuno-expression between the ipsi and contralateral trigeminal ganglion for sham, 4, 7 and 14 days post CCI. Ganglia from sham-operated rats (E, standard, G, thresholded image) showed no difference compared to contralateral ganglia (F, standard, H, thresholded image). J, white bars show total glutamate immunopositive cells sorted by size from 9 sections of three 14-day CCI-ION trigeminal ganglion and the black bars show the number glutamate positive cells above threshold. Scale bar = 50 µm *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 9.
Relation between injured neurons and change in glutamate expression.
Seven days following CCI of the inferior orbital nerve many neurons show increased levels of glutamate immunosignal (A, arrows and see Figure 8). Not all these cells are double labeled for ATF3 (B, arrows) a marker of neuronal injury. The neurons indicated with arrowheads are also above threshold but not ATF3 immuno positive. This is not due to the nucleus being out of the plane of section as DAPI staining (C, arrowheads) shows nuclei are present. Scale bar = 30 µm.