Endogenous peptidergic modulation of perisynaptic Schwann cells at the frog neuromuscular junction

Abstract

1. Although peptides are important modulators of synapses, their action on synapse-glia interactions remain unclear. The amphibian neuromuscular junction (NMJ) was used to examine the effects of substance P (SP) on perisynaptic Schwann cells (PSCs), glial cells at the frog NMJ, by monitoring changes in intracellular Ca2+. 2. SP induced Ca2+ responses that were mimicked by the neurokinin 1 receptor (NK-1) agonist septide and with a shorter delay by the SP fragment, SP(6-11). SP and SP(6-11) responses were blocked by NK-1 antagonists SR140333 and LY303870. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed but were blocked after pertussis toxin (PTX) treatment, indicating that the receptors were linked to internal stores of Ca2+ via a PTX-sensitive G-protein. 4. The slowly hydrolysable NK-1 agonist [Sar9, Met(O2)11]-SP only induced Ca2+ responses when applied for a long period of time and not during brief, local applications, suggesting the involvement of SP hydrolysis. Acetylcholinesterase (AChE) may not be involved in SP degradation since Ca2+ responses evoked by SP were unchanged in the presence of the cholinesterase inhibitor neostigmine. 5. Ca2+ responses induced by muscarine and nerve stimulations were almost abolished when preceded by SP applications, while those induced by ATP were significantly reduced. The rundown of the nerve-evoked Ca2+ responses in PSCs was attenuated in the presence of SR140333. 6. These results indicate that endogenous SP is involved in the regulation of PSC activity and that SP is an important modulator of glial cell Ca2+ signalling and synapse-glia communication.

Figure 1. SP-induced Ca²⁺ responses in PSCs at frog NMJ

A, confocal images of Ca²⁺ responses to local applications of SP on PSCs. Relative fluo-3 fluorescence appears on a grey scale, where black corresponds to lower, and white corresponds to higher fluorescence. A1, three PSCs at rest; A2, during the Ca²⁺ response to local applications of 200 μm SP; A3, during the recovery period. The image of the responding PSCs does not necessarily show their peak responses since the onset was different for each cell. B, changes in cell body fluorescence (%ΔF/F) over time before, during and after the local drug applications (arrow) on the cell indicated by the arrow in A. Numbers beside the trace indicate the respective times at which images in A were taken. Note that a rebound in fluorescence intensity occurs during the recovery period. C, Ca²⁺ responses in PSCs evoked by a bath application (2 min) of SP (1 μm). One PSC is shown before (Rest) and at the peak of the Ca²⁺ response (SP). Scale bars, 10 μm.

Figure 2. SP(6–11)- and septide-induced Ca²⁺ responses in PSCs at frog NMJ

A, Ca²⁺ responses in PSCs evoked by local applications of SP(6–11). Images show 4 cells before (A1) and after local applications of 20 μm SP(6–11) (A2), and during recovery following drug applications (A3). The images of responding PSCs do not necessarily show their peak responses since the onset was different for each cell. B, time course of the Ca²⁺ response in the cell indicated by the arrow in A. Numbers beside the trace indicate the times at which the images in A were taken. No oscillations were observed during the recovery period. C, delays for Ca²⁺ changes induced by local applications (arrow) of SP and SP(6–11) on the same PSC. Each trace illustrates the time course of Ca²⁺ responses for the first 45 s after the agonist applications, with an interval of 10 min between the two applications. 20 μm SP(6–11) (○) induced Ca²⁺ responses after 20.3 s and 200 μm SP (•) induced Ca²⁺ responses after 33.9 s. D, confocal images of Ca²⁺ responses to local applications of septide (20 μm), a SP agonist specific for NK-1 receptors. Images show two PSCs before (Control) and at the peak of the Ca²⁺ response evoked by local applications of septide (20 μm). Scale bars, 10 μm.

Figure 3. Effects of an NK-1 blocker on Ca²⁺ responses induced by SP and SP(6–11)

A, time course of the Ca²⁺ changes for 3 PSCs in response to local applications of agonists (arrows) in the presence of the NK-1 receptor blocker, SR140333. Local applications of 20 μm SP(6–11) failed to induce Ca²⁺ responses in PSCs with SR140333 (4 μm) in the perfusate. After washout of the first agonist (first break in the x-axis), local applications of 200 μm SP also failed to induce Ca²⁺ responses in the presence of SR140333. However, 10 min later (second break in the x-axis), Ca²⁺ responses could be induced with 10 μm muscarine, indicating the specific action of SR140333 on NK-1 receptors. B, confocal images of the Ca²⁺ fluorescence in two PSCs in the presence of SR140333 (Rest), during application of SP (200 μm) in the presence of SR140333 (SP/SR140333) and after the washout of SR140333 (SP). Note that the effects of SR140333 are reversible. Scale bar, 10 μm.

Figure 6. Modulation by SP of muscarine and ATP-induced Ca²⁺ responses in PSCs

A, percentage of responding cells and the mean of Ca²⁺ response in PSCs induced by local applications of 10 μm muscarine (□) and induced by local applications of muscarine (10 μm) following SP applications (20 μm; 10 min intervals; ▪). Note that both the percentage of responding cells and the size of muscarine-induced Ca²⁺ responses were significantly reduced after SP applications. B, percentage of responding cells and the mean of Ca²⁺ responses in PSCs evoked by local application of ATP (□) and evoked by ATP following local applications of SP (▪). Following applications of 20 μm SP (10 min intervals) the percentage of cells responsive to 20 μm ATP was not affected, whereas the mean Ca²⁺ response was significantly different from the control. Numbers of cells tested are shown in parentheses. C, normalized increase of fluorescence expressed as a percentage of the Ca²⁺ response in PSCs evoked by the first application of ATP. On average, consecutive applications of 10 μm ATP (10 min intervals) induced Ca²⁺ responses of similar amplitude (6 cells). D, normalized increase of fluorescence expressed as a percentage of the Ca²⁺ response in PSCs evoked by the first application of ATP after SP (500 nM) was perfused for 10 min (bar), immediately after the first ATP application. Note that a rundown in the mean Ca²⁺ response induced by 10 μm ATP was observed (6 cells, different preparation from C). Significant differences from the control values are shown by asterisks: * P = 0.02, ** P = 0.005, Student's t test.

Figure 4. Cellular mechanisms of SP-evoked Ca²⁺ responses in PSCs

Confocal images of Ca²⁺ responses in PSCs to local applications of SP. A, images of 2 PSCs incubated for 14 h in a PTX solution (2 μg ml⁻¹) before (Rest) and 25 s after local applications of SP (200 μm). Note the absence of Ca²⁺ responses in these conditions. However, Ca²⁺ responses could be induced by muscarine (10 μm) in the same cells. B, images showing 2 PSCs, from a different preparation to A, at resting level (Rest) and 33 s after local applications of SP following 20 min of perfusion with Mg²⁺ Ringer solution. Ca²⁺ responses were still induced by SP when no Ca²⁺ was added to the saline. Scale bars, 10 μm.

Figure 5. SP is active through a degradation product

A, confocal images of Ca²⁺ responses to a bath application (2 min) of [Sar⁹, Met(O₂)¹¹]-SP, a SP analogue more resistant to enzymatic degradation. Images show two cells before (Control) and at the peak of the Ca²⁺ response induced by [Sar⁹, Met(O₂)¹¹]-SP (200 μm) which occurred after 120 s, B, Ca²⁺ responses to local applications of [Sar⁹, Met(O₂)¹¹]-SP. Images show one cell before (Rest), 20 s after local applications of [Sar⁹, Met(O₂)¹¹]-SP (200 μm), and at the peak of the Ca²⁺ response evoked by local applications of SP (200 μm). C, Ca²⁺ responses obtained by local applications of SP, 20 min after addition of 3 μg ml⁻¹ neostigmine. Images show 2 cells before (Rest) and 35 s after local applications of SP (200 μm). Note that Ca²⁺ responses were still evoked by SP in the presence of neostigmine. Scale bars, 10 μm.

Figure 7. Endogenous SP modulates nerve-evoked Ca²⁺ signals in PSCs

A, Ca²⁺ changes in PSCs over time before, during (bar), and after release of transmitter induced by repetitive nerve stimulation (50 Hz, 30 s). In control (continuous trace), nerve-evoked Ca²⁺ responses were characterized by progressive rundown during successive stimulations at intervals of 25 min (data from second and third trains not shown). Often, no Ca²⁺ response could be evoked by a fourth train. However, in the presence of the NK-1 blocker SR140333 (1 μm) (dotted trace) the rundown of nerve-evoked Ca²⁺ responses was less pronounced (data from second and third trains not shown). In some cases, oscillations were also observed. B, mean Ca²⁺ responses evoked by repetitive trains of stimuli to the motor nerve (50 Hz, 30 s; at intervals of 25 min) for controls (□) and cells in the presence of SR140333 (▪). Note the persistence of the Ca²⁺ response evoked by the third and fourth train of stimuli in the presence of SR140333. Significant differences from the control values are indicated by asterisks: * P = 0.02, ** P = 0.005, Student's t test. C, changes in fluo-3 fluorescence over time in 3 PSCs (arrowheads) induced by repetitive nerve stimulation (50 Hz, 30 s) to evoke the release of neurotransmitters. Ca²⁺ responses are associated with their respective PSCs by an arrow. SP (20 μm) was locally applied to the top cell. Scale bar, 10 μm.