Synaptic plasticity in myenteric neurons of the guinea-pig distal colon: presynaptic mechanisms of inflammation-induced synaptic facilitation

Abstract

The purpose of this study was to investigate the pre- and postsynaptic mechanisms that contribute to synaptic facilitation in the myenteric plexus of the trinitrobenzene sulphonic acid-inflamed guinea-pig distal colon. Intracellular recordings of evoked fast excitatory postsynaptic potentials (fEPSPs) in myenteric S neurons were evaluated, and the density of synaptic terminals was morphometrically analysed by transmission electron microscopy. In inflamed tissue, fEPSPs were reduced to control levels by the protein kinase A (PKA) inhibitor, H89, but H89 did not affect the fEPSPs in control tissue. This PKA activation in inflamed tissue did not appear to involve 5-HT(4) receptors because the antagonist/inverse agonist, GR 125487, caused comparable decreases of fEPSPs in both tissues. Inhibition of BK channels with iberiotoxin did not alter the fEPSPs in inflamed tissue, but increased the fEPSPs in control tissue to the amplitude detected in inflamed tissue. During trains of stimuli, run-down of EPSPs was less extensive in inflamed tissue and there was a significant increase in the paired pulse ratio. Depolarizations in response to exogenous neurotransmitters were not altered in inflamed tissue. These inflammation-induced changes were not accompanied by alterations in the pharmacological profile of EPSPs, and no changes in synaptic density were detected by electron microscopy. Collectively, these data indicate that synaptic facilitation in the inflamed myenteric plexus involves a presynaptic increase in PKA activity, possibly involving an inhibition of BK channels, and an increase in the readily releasable pool of synaptic vesicles.

Figure 1. The pharmacological profiles of myenteric fEPSPs are not altered in the inflamed tissue

A, bar graph illustrating the percentage of total fEPSPs that are purely nicotinic, nicotinic/purinergic, nicotinic/purinergic/other, or nicotinic/other in control (open bars) and inflamed colons (grey bars) with n values of each type within each bar. B, mean +s.e.m. representing the percentage contribution of individual neurotransmitters to the total amplitude of mixed EPSPs in control (open bars) and inflamed colons (grey bars; n = 21 for control, n = 19 for inflamed; P = 0.63, χ² analysis). C, representative traces from control tissue of the four types of fEPSPs from the myenteric plexus: (1) nicotinic; (2) nicotinic/purinergic; (3) nicotinic/purinergic/other; and (4) nicotinic/other. Dashed lines represents baseline of EPSP amplitude. All traces are on the same voltage and time scale. Bold arrows are pointing at the stimulus artifact.

Figure 2. The PKA inhibitor, H89, attenuates EPSPs in inflamed, but not control, tissue

A, mean +s.e.m. of the fEPSP amplitudes from control (open bars) and inflamed colons (grey bars) in Krebs solution and in Krebs solution with protein kinase inhibitor H89 (10 μm; n = 5 for control, n = 6 for inflamed; *P < 0.05 compared with before H89, Student's paired t test). B, representative traces illustrating the differences in amplitudes and sensitivity of fEPSPs to H89 from control and inflamed colons. Traces are on the same voltage and time scales. Bold arrows are pointing to the stimulus artifact.

Figure 3. Inflamed tissue is less sensitive to forskolin than control tissue

A, mean +s.e.m. of the fEPSP amplitudes from control (open bars) and inflamed colons (grey bars) in Krebs solution and in Krebs solution with the adenylate cyclase activator, forskolin (10 μm; n = 5 for both control and inflamed; *P < 0.05 compared with before forskolin, Student's paired t test; †P < 0.05 different from control, Student's unpaired t test). B, representative traces illustrating the difference in amplitudes and sensitivities of fEPSPs from control and inflamed colons. Traces are on the same voltage and time scales. Bold arrows are pointing to the stimulus artifact.

Figure 4. The 5-HT₄ receptor antagonist/inverse agonist, GR 125487, causes a slight inhibition of the fast EPSP in both control and inflamed tissue

Mean +s.e.m. of the fEPSP amplitudes from control (open bars) and inflamed colons (grey bars) in Krebs solution and in Krebs solution with the 5-HT₄ receptor antagonist/inverse agonist, GR 125487 (100 nm; n = 7 for control, n = 10 for inflamed; †P < 0.05 different from control animals, Student's unpaired t test for Krebs data, and two-way ANOVA with repeated measures for GR 125487 data; *P < 0.05 compared with before GR 125487, Student's paired t test).

Figure 5. The BK channel blocker, iberiotoxin, facilitates the EPSP in control, but not inflamed, tissue

A, mean +s.e.m. of the fEPSP amplitudes from control (open bars) and inflamed colons (grey bars) in Krebs solution and in Krebs solution with the BK channel blocker, iberiotoxin (100 nm; n = 5 for both control and inflamed; *P < 0.05 compared with before iberiotoxin, Student's paired t test; †P < 0.05 different from control, Student's unpaired t test). B, representative traces illustrating the difference in amplitudes and sensitivities of fEPSPS from control and inflamed colons. Traces are on the same voltage and time scales. Bold arrows are pointing to the stimulus artifact.

Figure 6. Synaptic run-down is less extensive in inflamed tissue than in control tissue

A, mean ±s.e.m. of the percentage change in amplitudes of fEPSPs compared with the first amplitude in control (open squares) and inflamed colons (grey triangles) at 0.5, 5, 10 and 20 Hz. Note how the run-down properties appear to change at 5 Hz, but a significant difference in run-down between control and inflamed tissues was only detected at 20 Hz (0.5 Hz, n = 15 for control, n = 16 for inflamed; 5 Hz, n = 13 for both control and inflamed; 10 Hz, n = 11 for control, n = 13 for inflamed; and 20 Hz, n = 11 for control, n = 13 for inflamed; 20 Hz, P < 0.05, two-way ANOVA with repeated measures). B, representative traces of the first 10 evoked fEPSPs during a train stimulation, illustrating the difference in run-down between control and inflamed colons at 20 Hz. Both traces are on the same voltage and time scales. Each bold arrow is pointing to stimulus artifact.

Figure 7. Inflammation causes an increase in the paired pulse ratio

A, mean +s.e.m. of the ratio of the second fEPSP to the first fEPSP at the 50 ms latency from control (open bars) and inflamed colons (grey bars; (*P < 0.05 different from control, Student's unpaired t test). B, representative traces illustrating PPD in control and no change in the EPSP amplitudes in the inflamed colons in response to pairs of stimuli. Traces are on the same voltage and time scales. Bold arrows indicate the stimulus artifact.

Figure 8. Exogenous neurotransmitters evoke comparable depolarizations in neurons from control and inflamed preparations

A, bar graphs of mean +s.e.m. illustrating the fEPSP amplitudes for all neurons in the study and the maximum amplitude of response in S neurons in response to exogenously applied neurotransmitters ACh or ATP in control (open bar) and in inflamed colons (grey bar). (fEPSPs amplitudes, n = 93 for control, n = 95 for inflamed, P < 0.0001; ACh, n = 12 for control, n = 9 for inflamed; ATP, n = 10 for both control and inflamed, P > 0.1, Student's unpaired t test.) B, representative traces from control (top) and inflamed colons (bottom), with the synaptic response in the first column and the response to exogenously applied neurotransmitters ACh (second column) and ATP (third column). The time and voltage scales are displayed for each trace. Bold arrows point to stimulus artifact in synaptic response trace and point of neurotransmitter application in response to transmitter traces.

Figure 9. No change in synaptic contact density is observed in the inflamed tissue

A, bar graphs of mean +s.e.m. of the percentage of postsynaptic membrane directly apposing presynaptic terminals, number of synaptic contacts per micrometre and average area of all discernable nerve terminals in control (open bar) and inflamed colons (grey bar; number of contacts, n = 15 for control, n = 16 for inflamed; percentage membrane covered, n = 15 for control, n = 16 for inflamed; and nerve terminal area, n = 8 for control, n = 14 for inflamed; P > 0.2, Student's unpaired t test). B, electron micrograph of a representative nerve terminal (T) that is contacting a neuron in the field, and a terminal (t) that is not considered to be contacting the postsynaptic neuron. The nerve terminal is circled to illustrate how terminal area was measured. The rectangle denotes the region of the postsynaptic neuronal membrane that is directly apposing the nerve terminal, and the arrows are pointing to the neuronal cell membrane. Scale bar represents 500 nm.