Divergent effects of painful nerve injury on mitochondrial Ca(2+) buffering in axotomized and adjacent sensory neurons

Abstract

Mitochondria critically regulate cytoplasmic Ca(2+) concentration ([Ca(2+)]c), but the effects of sensory neuron injury have not been examined. Using FCCP (1µM) to eliminate mitochondrial Ca(2+) uptake combined with oligomycin (10µM) to prevent ATP depletion, we first identified features of depolarization-induced neuronal [Ca(2+)]c transients that are sensitive to blockade of mitochondrial Ca(2+) buffering in order to assess mitochondrial contributions to [Ca(2+)]c regulation. This established the loss of a shoulder during the recovery of the depolarization (K(+))-induced transient, increased transient peak and area, and elevated shoulder level as evidence of diminished mitochondrial Ca(2+) buffering. We then examined transients in Control neurons and neurons from the 4th lumbar (L4) and 5th lumbar (L5) dorsal root ganglia after L5 spinal nerve ligation (SNL). The SNL L4 neurons showed decreased transient peak and area compared to control neurons, while the SNL L5 neurons showed increased shoulder level. Additionally, SNL L4 neurons developed shoulders following transients with lower peaks than Control neurons. Application of FCCP plus oligomycin elevated resting [Ca(2+)]c in SNL L4 neurons more than in Control neurons. Whereas application of FCCP plus oligomycin 2s after neuronal depolarization initiated mitochondrial Ca(2+) release in most Control and SNL L4 neurons, this usually failed to release mitochondrial Ca(2+) from SNL L5 neurons. For comparable cytoplasmic Ca(2+) loads, the releasable mitochondrial Ca(2+) in SNL L5 neurons was less than Control while it was increased in SNL L4 neurons. These findings show diminished mitochondrial Ca(2+) buffering in axotomized SNL L5 neurons but enhanced Ca(2+) buffering by neurons in adjacent SNL L4 neurons.

Keywords: Mitochondrial Ca(2+); Neuropathic pain; Sensory neuron function; Spinal nerve ligation.

Figure 1

Measures and protocol validation. (A) Measured parameters for K⁺-induced Ca²⁺ transients are shown for resting level, amplitude, peak, shoulder level, and time for 80% recovery to resting level. (B) To determine shoulder level, [Ca²⁺]_c traces (top panel) were differentiated (bottom panel, with the first positive and first negative maxima clipped), and the presence of two negative maxima (indicated by *) was considered as evidence of a shoulder. The level of the shoulder was measured at the inflection point (dashed arrows), identified as time during the recovery phase at which the slope of the [Ca²⁺]_c vs. time trace transient shifted from becoming less negative to becoming more negative. (C) To test adequacy of ATP levels, plasma membrane Ca²⁺ ATPase (PMCA) activity is measured as the time constant (τ) for the exponential recovery of [Ca²⁺]_c from small Ca²⁺ loads. The top panel is an example of repeated depolarization during application of Tyrode’s solution (Tyr) and after vehicle application in Tyrode’s. The bottom panel shows response to co-administration of FCCP (1μM) and oligomycin (10μM). (D) Average data show PMCA activity is unchanged. (E) Application of FCCP successfully reduces mitochondrial ΔΨ_m recorded by TMRM fluorescence, normalized as percent of baseline.

Figure 2

Demonstration traces of the response of neuronal [Ca²⁺]_c to depolarizations induced by K⁺ application of 0.5s and 1.3s duration in Tyrode’s solution (Tyr) and 1.5 minutes after switching to a bath solution containing either vehicle (A) or FCCP/Oligo (B). Response to FCCP/Oligo application is shown for neurons from Control dorsal root ganglion and from the fourth lumbar (L4) and L5 ganglia after L5 spinal nerve ligation (SNL). The second depolarization was initiated only after recovery from the preceding depolarization. During FCCP/Oligo application, the amplitude of the 1.3s transient increases by 2.10-fold for Control, 2.25-fold for SNL L4, and 1.53-fold for SNL L5, compared to baseline (Tyr).

Figure 3

Repeated depolarization protocol and determination of Ca²⁺ load adequate to produce a shoulder. (A) Cytoplasmic Ca²⁺ concentration trace (top) and its differentiation trace (bottom) in a control neuron, showing the protocol for sequential depolarizations, and the identification of a shoulder (indicated by dashed arrows) during the recovery phase of transients with larger Ca²⁺ loads. (B) Determination for each neuron of the lowest peak associated with the appearance of a shoulder shows that neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4, n=24) developed shoulders associated with lower transient peaks than control (C, n=50) or SNL L5 (n=71) groups. This and other boxplots show median and first and third quartiles (Q₁, Q₃) with whiskers denoting the lowest datum still within 1.5 times the interquartile range of Q₁ and the highest datum still within 1.5 times the interquartile range of Q₃*P < 0.05.

Figure 4

Comparison of FCCP/Oligo-sensitive dimensions of transients induced by 1.3s K⁺ depolarization in Control neurons compared to neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4) and SNL L5. (A) Determination of the transient peak. (B) Determination of the transient area. (C) Determination of the level of the shoulder during transient recovery. C, n=56; SNL L4, n=28; SNL L5, n=77. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Effect of nerve injury on mitochondrial regulation of resting [Ca²⁺]_c. (A) Resting [Ca²⁺]_c in Control neurons (C, n=279), neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4, n=76), and SNL L5 (n=113). (B) Sample traces showing the effect of co-administration of FCCP (1μM) and oligomycin (10μM, FCCP/Oligo) after application of Tyrode’s solution (Tyr). (C) Summary data comparing the response of resting [Ca²⁺]_c in neurons from Control animals to vehicle (Veh, n=63), and the response to FCCP/Oligo of neurons from Control (C, n=94), SNL L4 (n=38) and SNL L5 (n=25) ganglia. **P < 0.01, ***P < 0.001.

Figure 6

Effect of nerve injury on mitochondrial Ca²⁺ stores following neuronal activation. (A) Sample traces from control neurons, neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4), and SNL L5, showing response of [Ca²⁺]_c to K⁺ depolarization (5s) and to application of FCCP (1μM) and oligomycin (10μM, FCCP/Oligo) following bath superfusion of Tyrode’s solution. (B) The left panel shows the frequency of measurable Ca²⁺ release triggered by application of FCCP/Oligo to uninjured neurons bathed either in normal Tyrode’s solution (n=46) or to matched neurons in which the bath was switched to Ca²⁺-free Tyrode’s solution 5s before the application of FCCP/Oligo (in Ca²⁺-free vehicle) (n=36). The right panel shows amplitude of the [Ca²⁺]_c transient induced by the application of FCCP/Oligo, excluding data from neurons in with no measurable Ca²⁺ release. In both cases, there was no difference between groups normal Ca²⁺ bath (n=34) and Ca²⁺-free conditions (n=27). (C) The left panel shows summary data for the frequency of measurable Ca²⁺ release triggered by application of FCCP/Oligo to control neurons (C, n=135), SNL L4 (n=52), and SNL L5 (n=52). The right panel shows amplitude of the [Ca²⁺]_c transient induced by the application of FCCP/Oligo, excluding data from neurons in with no measurable Ca²⁺ release. Groups include control neurons (C, n=95), neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4, n=45), and SNL L5 (n=14). ***P < 0.001. (D) Peak of the [Ca²⁺]_c transient induced by application of K⁺ (5s). Groups include control neurons (C, n=305), neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4, n=52), and SNL L5 (n=52). ***P < 0.001. (E) Relationships between the peak of the K⁺-induced transient and the FCCP/Oligo-induced transient for each neuron, separately considered by group. Groups include control neurons (C, n=135), neurons from the fourth lumbar dorsal root ganglion after spinal nerve ligation (SNL L4, n=52), and SNL L5 (n=52). Data points for neurons with no measurable Ca²⁺ release (0 value for Y-axis) are displayed as a group below the plot, separated vertically for clarity. The regression lines represent a linear fit for the log transform of all data in that group, including non-responding neurons, for which the value of 5 (half of the lowest non-zero value in the data set) was substituted for the purpose of log transform and fitting the regression line. Comparison of the regression lines showed that the slope for SNL L4 was greater than for SNL L5 (P < 0.05), the Y-intercept for SNL L4 was greater than for Control (P < 0.01), and the Y-intercept for Control was greater than for SNL L5 (P <0.001).

Figure 7

A summary of possible underlying functional changes that could explain nerve injury effects on mitochondrial function. Cycling of Ca²⁺ through the mitochondria is illustrated as uptake by the Ca²⁺ uniporter and release by the Na⁺/Ca²⁺ exchanger. Axotomized neurons accumulate less Ca²⁺ in the mitochondrial matrix and retain Ca²⁺ less well. In contrast, the adjacent neurons that are intact but share peripheral nerve fascicles with degenerating segments of the axotomized neurons develop exaggerated Ca²⁺ accumulation without evidence of an altered release process.