Stimulation of Glia Reveals Modulation of Mammalian Spinal Motor Networks by Adenosine

Abstract

Despite considerable evidence that glia can release modulators to influence the excitability of neighbouring neurons, the importance of gliotransmission for the operation of neural networks and in shaping behaviour remains controversial. Here we characterise the contribution of glia to the modulation of the mammalian spinal central pattern generator for locomotion, the output of which is directly relatable to a defined behaviour. Glia were stimulated by specific activation of protease-activated receptor-1 (PAR1), an endogenous G-protein coupled receptor preferentially expressed by spinal glia during ongoing activity of the spinal central pattern generator for locomotion. Selective activation of PAR1 by the agonist TFLLR resulted in a reversible reduction in the frequency of locomotor-related bursting recorded from ventral roots of spinal cord preparations isolated from neonatal mice. In the presence of the gliotoxins methionine sulfoximine or fluoroacetate, TFLLR had no effect, confirming the specificity of PAR1 activation to glia. The modulation of burst frequency upon PAR1 activation was blocked by the non-selective adenosine-receptor antagonist theophylline and by the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, but not by the A2A-receptor antagonist SCH5826, indicating production of extracellular adenosine upon glial stimulation, followed by A1-receptor mediated inhibition of neuronal activity. Modulation of network output following glial stimulation was also blocked by the ectonucleotidase inhibitor ARL67156, indicating glial release of ATP and its subsequent degradation to adenosine rather than direct release of adenosine. Glial stimulation had no effect on rhythmic activity recorded following blockade of inhibitory transmission, suggesting that glial cell-derived adenosine acts via inhibitory circuit components to modulate locomotor-related output. Finally, the modulation of network output by endogenous adenosine was found to scale with the frequency of network activity, implying activity-dependent release of adenosine. Together, these data indicate that glia play an active role in the modulation of mammalian locomotor networks, providing negative feedback control that may stabilise network activity.

Fig 1. PAR1 immunoreactivity co-localises with GFAP but not with MAP2 in the lumbar ventral spinal cord.

A: representative images showing 50 μm transverse sections taken from the upper lumbar spinal cord of a P6 C57BL/6 mouse. Sections were stained with antibodies raised against GFAP (green) and PAR1 (red). B: representative images showing 50 μm transverse sections taken from the upper lumbar spinal cord of a mouse. Sections were stained with antibodies raised against MAP2 (green) and PAR1 (red). Arrows in Bi indicate areas of PAR1 staining between MAP2+ cells. Scale bars: 20 μm.

Fig 2. Stimulation of glia during ongoing locomotor-related activity results in a transient reduction in burst frequency.

A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots and the right L₅ ventral root showing the effect of the PAR1 agonist TFLLR (10 μM). Bi: locomotor-burst frequency over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period. Individual data points are shown in grey and means are represented by black lines. Bii: time course plot of normalised data aggregated into 1-min bins showing a reduction in burst frequency upon application of TFLLR. Ci: locomotor-burst amplitude over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period. Cii: time course plot of normalised data aggregated into 1-min bins showing no change in burst amplitude upon application of TFLLR. n = 10 preparations. Statistically significant differences in pairwise comparisons: *p < 0.05, **p < 0.01.

Fig 3. TFLLR has no effect on locomotor-related bursting following pharmacological ablation of glia.

A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) following glial ablation with methionine sulfoximine (MSO; 100 μM), which was co-applied with glutamine (Gln; 1.5 mM). B: locomotor-burst frequency in the presence of MSO and Gln over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period. Individual data points are shown in grey and means are represented by black lines. n = 10.

Fig 4. Glial stimulation results in release of adenosine and activation of neuronal A₁ receptors.

A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) applied in the presence of theophylline (20 μM). B: locomotor-burst frequency in the presence of the non-selective adenosine receptor antagonist theophylline over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). C: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) applied in the presence of the A₁-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 50 μM). D: locomotor-burst frequency in the presence of DPCPX over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). E: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) applied in the presence of SCH58261 (25 μM). F: locomotor-burst frequency in the presence of the A_2A-receptor antagonist SCH58261 over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 10). Individual data points are shown in grey and means are represented by black lines. Statistically significant differences in pairwise comparisons: *p < 0.05.

Fig 5. Modulation of locomotor network output upon glial stimulation requires extracellular degradation of ATP to adenosine.

A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) applied in the presence of the ectonucleotidase inhibitor ARL67156 (50 μM). B: locomotor-burst frequency in the presence of ARL67156 (50 μM) over 3 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 11).

Fig 6. ATP-adenosine released following stimulation of glia modulates inhibitory components of locomotor networks.

A: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the PAR1 agonist TFLLR (10 μM) applied to preparations in which inhibitory transmission was blocked by the GABA_A-receptor antagonist pictrotoxin (10 μM) and the glycine-receptor antagonist strychnine (1 μM). B: locomotor-burst frequency in disinhibited preparations over 6 min during a control period, immediately following TFLLR application, and following a 20-min washout period (n = 13). Individual data points are shown in grey and means are represented by black lines.

Fig 7. Adenosinergic modulation of locomotor networks scales with network activity.

A-C: raw (top) and rectified/integrated (bottom) traces recorded from left (L) and right (R) L₂ ventral roots showing the effect of the A₁-receptor antagonist DPCPX (50 μM) in preparations in which fictive locomotion was evoked by 5-HT (10 μM) and DA (50 μM) alone (A) or with 3 μM NMDA (B) or 5 μM NMDA (C). D: percentage change in locomotor-burst frequency in response to DPCPX application in preparations in which fictive locomotion was evoked at different frequencies using 0 μM NMDA (n = 11), 3 μM NMDA (n = 16) and 5 μM NMDA (n = 17), calculated by comparing a 10-min control period with the last 10 min of a 30-min application of DPCPX. Individual data points are shown in grey and means are represented by black lines. Statistically significant differences in pairwise comparisons: *p < 0.05, ***p < 0.001.