Organization of sensory input to the nociceptive-specific cutaneous trunk muscle reflex in rat, an effective experimental system for examining nociception and plasticity

Abstract

Detailed characterization of neural circuitries furthers our understanding of how nervous systems perform specific functions and allows the use of those systems to test hypotheses. We have characterized the sensory input to the cutaneous trunk muscle (CTM; also cutaneus trunci [rat] or cutaneus maximus [mouse]) reflex (CTMR), which manifests as a puckering of the dorsal thoracolumbar skin and is selectively driven by noxious stimuli. CTM electromyography and neurogram recordings in naïve rats revealed that CTMR responses were elicited by natural stimuli and electrical stimulation of all segments from C4 to L6, a much greater extent of segmental drive to the CTMR than previously described. Stimulation of some subcutaneous paraspinal tissue can also elicit this reflex. Using a selective neurotoxin, we also demonstrate differential drive of the CTMR by trkA-expressing and nonexpressing small-diameter afferents. These observations highlight aspects of the organization of the CTMR system that make it attractive for studies of nociception and anesthesiology and plasticity of primary afferents, motoneurons, and the propriospinal system. We use the CTMR system to demonstrate qualitatively and quantitatively that experimental pharmacological treatments can be compared with controls applied either to the contralateral side or to another segment, with the remaining segments providing controls for systemic or other treatment effects. These data indicate the potential for using the CTMR system as both an invasive and a noninvasive quantitative assessment tool providing improved statistical power and reduced animal use.

Keywords: anesthesiology; animal models; pain; pharmacology; plasticity; sensory neurons; spinal cord.

Figure 1

Gross dissection of the CTM neural supply (Lateral Thoracic nerve; LTn) emerging from the brachial plexus and branching to run through the muscle (arrows). View is of the left side of the rat, head at top left, with skin incised at midline and reflected laterally (left). The dashed line demarcates the edge of the CTM.

Figure 2

Schematic drawing of dorsal cutaneous nerves with some distinguishing landmarks.

Figure 3

The proportion of animals in which the CTMR can be observed either by LTn neurogram (black squares) or CTM EMG (gray triangles) in response to electrical stimulation of segmental dorsal cutaneous nerves is highest from C7-L4. Stimuli were single pulses at C-fiber strength (3-5mA amplitude, 0.5ms duration).

Figure 4

Neurogram recordings reveal that the CTMR is weaker following stimulation of segments at the rostral and caudal borders of sensory input (C7-8 and L5-6, respectively) than from main sensory input segments (T1-L4), represented by the response from stimulating the T10 DCn. Traces are averages of 20 responses.

Figure 5

The CTMR is visible in response to pinch stimulation over a large area of dorsal skin. Black dots indicate sites of light pinch that induced a CTMR. Some dots were digitally enhanced.

Figure 6

A CTMR EMG can be recorded in response to different types of stimuli including skin pinch (A), tap of subcutaneous superficial fascia over vertebral spinous process (B), or electrical stimulation of L3 DCn (C). Application of pinch (A) and tap (B) are indicated as bars above the EMG traces. (A) and (B) are single sweeps and (C) is the average of 20 sweeps (each sweep was from a single stimulus at C-fiber strength). Duration of (A) is 2.5s, (B) is 500ms, and (C) is 250ms.

Figure 7

Pinch of this subcutaneous aponeurosis tissue induced a CTMR. Pinch-marks (black arrowheads) cross axon(s) (white arrows) in a section of. Scale bar indicates 10 microns.

Figure 8

Elimination of p75-bearing neurons affects the ability of cutaneous sensory nerves to drive a CTMR. A) Electrophysiological measurement of the CTMR from an animal that received an injection of 192-saporin into the left T13 DRG. Traces are averaged neurogram recordings (20 sweeps) from the lateral thoracic nerve (LTn) in response to electrical stimulation (current intensity indicated to left) of various DCnn (indicated below traces). The neurograms also reveal motion artifact (*) due to CTMR-induced contraction of the muscle which occurred because the non-recorded branches of the LTn were left intact to enable comparison of LTn motor neurogram and behavioral CTMR. The presence of a CTMR in response to stimulation of the L1 DCn indicates that the surgical procedures used to prepare the dorsal roots for recording did not damage the spinal cord. A-inset) Electrophysiological recording of the dorsal root compound action potential (average of 25 traces) in response to stimulation of the T13 DCn to confirm conduction in spared C-fiber primary afferents (arrow). B) Histochemical assessment of naive and 192-saporin-injected DRG for IB4-binding, and immunoreactivity for trkA, somatostatin (SOM), and P2X3. Scale bars indicate 25μm.

Figure 9

In Case 8 where the 192-sap toxin was only partially effective, killing nearly all A-fibers, but sparing C-fibers, the “earlier” CTMR component evoked in response to C-fiber-strength electrical stimulation of the DCn was selectively lost, while the “later” CTMR component remained. CTMR neurograms are shown in A (to stimulation of the control segment) and B ((to stimulation of the 192-sap-injected segment). Dorsal root recordings to the same stimulus parameters that evoked the CTMR responses in A and B are shown in C (A-fiber response), and D (C-fiber response; traces are overlaid to enable comparison). Traces from control segments are in gray, and from 192-sap-injected segments are in black. The scale bar indicates 10ms for A, B, and D, and 2ms for C, and 100mV for A and B, and 500mV for C and D.

Figure 10

Dexmedetomidine extends the anesthetic actions of bupivacaine assessed with CTMR induced by skin pinch (for behavioral assay) or electrical stimulation of the treated nerve (for EMG assay). (A) Dex-mediated extension of anesthetic duration over Bupivacaine alone is significant (p-values shown in bars); 26% when assessed behaviorally and 28.1% when assessed electrophysiologically. Bars represent mean ± standard deviation. (B) Scatterplot and regression analysis showing results from individual animals. Points of “no effect” of Dexmedetomidine are indicated by the dotted line. (C) Schematic timeline of the effects of Dexmedetomidine on the CTMR. (D) EMG recordings of the CTMR induced by electrical stimulation of the treated nerves. Time (minutes) relative to treatment is indicated to the left of the traces. The traces represent the baseline (-5 minutes) and the trial during which the reflex began to return on the Bupivacaine side (left panel; +150 min) and bupivacaine + dexmedetomidine side (right panel; +190 min).

Figure 11

The effect of dexmedetomidine, applied directly to the sciatic nerve in vivo, on nerve conduction was assessed by dorsal root recordings in response to stimulation of the peroneal nerve. Effect on the early component of the compound action potential (indicated by dotted bar) was rapidly reversed by the competitive alpha-2 antagonist atipamezole (Atip), administered iv. Effect on the later component of the CAP (indicated by open bar) was minimally reversed by Atip in this timeframe. Each sweep represents the average of 30 traces (delivered at 1Hz); arrow indicates stimulus artifact.

Figure 12

The effects of BDNF on spinal nociceptive processing were assessed with the CTMR. (A) The values for each parameter are plotted over time. Each point corresponds to a single trial (20 single pulses delivered at 0.9Hz, 0.5ms duration, 5mA amplitude). Arrow indicates the time at which aliquots (BDNF or aCSF) were added to the pools. (B) The neurogram traces (20 traces added and rectified) from the baseline trial and the final trial are presented. The inset shows overlaid traces magnified at the CTMR onset from the T12 segment before and after (arrowed) BDNF treatment to demonstrate the latency shift.

Figure 13

This schematic represents the basic proposed spinal circuitry of the CTMR, not including other sources of input to the CTM motoneurons such as those from brainstem, and some characteristics that may provide indications of different forms of plasticity. It is arranged as if the animal were prone with its head to the left and midline at the top of the image, with the skin reflected laterally (toward bottom of image), causing the CTM underlying the skin to be at the “top” of the skin and the epidermis to be at the “bottom”. Dotted lines represent neural elements or stimulus modalities that do not drive the reflex normally (dark large-diameter DRG neuron, LCn, DCn). T4, T10, and L2 segments were chosen to graphically indicate rostrocaudal progression and do not indicate that the schematized characteristics are present only at those segments. The lower panel represents some of the possible sites of plasticity and how the characteristics of the CTMR may change accordingly (numbered stars). Plasticity is noted by double lines not present in the top panel, and the conversion of dotted lines to solid lines.