Electrophysiologic Characteristics of Large Neurons in Dorsal Root Ganglia during Development and after Hindpaw Incision in the Rat

Abstract

Introduction

Withdrawal thresholds in the paw are lower in younger animals and incision further reduces these thresholds. We hypothesize these differences result in part from changes in intrinsic electrophysiologic properties of large neurons.

Methods

Using isolated whole dorsal root ganglion, current clamp was performed to determine the electrophysiologic properties of large neurons before and after incision in animals of one and four weeks of age. Mechanical withdrawal thresholds were used to follow paw sensitivity.

Results

Following paw incision, withdrawal thresholds decreased to a similar degree at both ages, but returned to control threshold at 72 hours only in the one week old. The resting membrane potential was less negative, the rheobase and the resistance of the membrane were lower at baseline in the one week old animals (p<0.05). After incision, the membrane potential became more depolarized and the rheobase was less in both ages. These changes remained 72 hours after the incision at both ages.

Conclusion

These findings suggest that lower mechanical thresholds in the younger animals may be partially attributed to the intrinsic electrophysiologic properties of the larger diameter afferent neurons. The lack of resolution of the electrophysiologic changes in the young despite the resolution of the withdrawal response suggests that continued input from large fibers into the central nervous system may occur at this age despite the apparent resolution of behavioral changes. Further studies are needed to determine the etiology of these differences, their impact in the central nervous system, and if theses changes can be prevented.

Introduction

Differences in response to mechanical stimulation in the periphery exist in both animals and in humans during development.^1–3 In the young the peripheral afferent nerve has a larger receptive field and is more excitable, with a larger response to a given stimulus, including long-lasting after discharges.^4,5 Additionally, spontaneous activity can occasionally be seen in the immature afferent fibers, a finding not commonly observed in later development unless the nerve or spinal cord is damaged.^4,6

Surgery is performed in patients of all ages, including extremes of immaturity. Surgical tissue trauma results in a cascade of events which culminate in spontaneous pain and hypersensitivity to mechanical stimuli.^2,7,8 Initially there is local activation of peripheral nerves from disruption of the integrity of the skin and transection injury to small nerve endings. This is followed by release of inflammatory mediators and further increase in neural signaling. In addition, healing is associated with hyperinnervation to the previously injured skin that is greater in the younger animal.⁷ These peripheral events occur simultaneously with changes in central processing of afferent input over time.⁹

After incision there is a more rapid resolution of hypersensitivity to mechanical stimuli in the young.² Different responses to the incision at the neuronal level may partially underlie short and long term effects of tissue injury. The signal from the local injury passes through the dorsal root ganglion (DRG) on its way to the dorsal horn of the spinal cord. Alterations in intrinsic excitability of DRG cells have been previously shown in nerve injury and in DRG compression.^10–12 The majority of the nerve cell’s transcriptional machinery resides here making this a crucial part of the peripheral nerve cell. Peripheral activity may induce changes and development may modulate this signaling. Examination of intracellular and membrane electrophysiology of peripheral afferents is possible at the cell body without dissociating cells, and state of excitability of this part of the neuron’s membrane may reflect changes at peripheral and central terminals.

Noxious and innocuous stimuli early in development can have potentially adverse effects.¹³ Repetitive innocuous stimuli can result in abnormal signaling. Innocuous sensation or light touch is primarily carried by large myelinated or A beta fibers and can be assessed by response to von Frey filaments.¹⁴ The large neuronal cells in the DRG have been shown to be large myelinated fibers by conduction velocity and likely represent light touch under normal circumstances.¹⁵ In the rat physiologic function of the C fibers is not fully established until 2 weeks postnatally, yet A beta fibers function normally.¹⁶ However, anatomically A beta fibers extend into more superficial laminae in the young, and input from these fibers may be activating dorsal horn structures normally reserved for noxious signaling.¹⁷ Although A beta fibers in the adult do not seem to play a large role in responses to incisional pain after surgery, this may be different in the young due to the altered anatomic connections.^17–19 Therefore, understanding of both the activation of the structures in the dorsal horn by A beta fibers, but also the activity of these neurons during development and in response to incision is important. With a better understanding of the peripheral sensitivity and activity of these neurons during development, targeting of these nerves to prevent adverse sequela of surgery early in life may prove to be beneficial by reducing the impact incision has in the spinal cord and brain.^13,20 In this study, we hypothesized that the electrophysiological nerve cell properties which govern neuronal excitability differ during development and following incision, and that these developmental differences parallel differences in the behavioral response to surgical incision at different stages of development.

Materials and Methods

Animal Behavior and Surgery

After approval from the Animal Care and Use Committee (Wake Forest University, Forsyth County, Winston-Salem, North Carolina) male Sprague-Dawley rats at one and four weeks of age were studied. One-week animals are pre-weanling, and four week animals are post-weanling. After baseline testing, all animals were anesthetized with 2% halothane in oxygen under spontaneous ventilation through a nose cone. As previously described^2,8 the plantar aspect of the left hindpaw was prepared in a sterile manner with a 10% povidone-iodine solution. A midline incision from the heel to the base of the toes was performed using a #11 blade using sterile technique. Rather than a fixed length incision, this created an incision of a fixed proportion of the size of the paw at different ages.² A small forceps was used to elevate the flexor tendon from the heel to the toes. The incision was closed with 5.0 nylon on an FS-2 needle using two inverted mattress sutures and the suture left in place. Control animals underwent anesthesia with prep, but no incision.

Mechanical Stimulation Testing

Animals were placed on a solid surface in a plastic cage. They were acclimated to the environment for 20 min prior to testing. Withdrawal to mechanical stimulation was assessed holding the animal with the hind feet resting on the solid surface with a temperature of 37°C and application of calibrated von Frey filaments to the dorsum of the foot until the filaments bent. The von Frey filaments used were 3.84, 4.08, 4.31, 4.56, 4.74, 4.93, 5.18, 5.46, 5.88, 6.10 corresponding to 0.5, 0.9, 1.7, 3.7, 5.5, 8.0, 12.4, 21.5, 53.0, and 84 g, respectively. This was done 3 times with a positive response determined by brisk withdrawal of the paw. The force resulting in withdrawal with a 50% probability was determined using the up-down method as previously described.²¹ Withdrawal threshold was determined before surgery and at 6 hours, 24 hours and 72 hours afterwards and in animals with no surgery at the same time points. All animals were included in the data analysis, and no animal in the study had a wound dehiscence or infection during the study.

Microelectrode intracellular recording from DRG

Animals underwent general anesthesia with halothane and spontaneous ventilation for removal of DRG. After surgical dissection, the left L₅ DRG was removed from 18 animals at each age. This was 6 animals at each time for control, 24 hours and 72 hours after incision. DRG were placed in a recording chamber and mounted on the stage of an upright microscope (BX50-WI, Olympus America, Inc., Center Valley, PA). A U-shaped stainless steel wire on which three to four fine nylons fibers spanned the two sides was used to gently hold the ganglion immersed at the bottom of the chamber. The chamber was continuously perfused with oxygenated artificial cerebrospinal fluid containing (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH₂PO₄, 24 NaHCO₃, 10 dextrose, 1.2 MgCl₂, and 1.2 CaCl₂ (pH = 7.3) at a rate of 2 ml/min, and the temperature was maintained at 37 ± 1°C as described previously.^11,22

DRG cells were visualized under differential interference contrast through a digital camera (Hamamatsu, Japan). Intracellular electrophysiological recordings were obtained from each neuron in the study with a sharp microelectrode filled with 2.5 M potassium acetate (pH = 7.2). Satisfactory recordings were obtained with electrodes of 50–80 MΩ. Before electrode penetration, the DRG soma was visually classified according to its diameter as large (≥45 μm). Cell size was measured directly from oscilloscope in 2 dimensions and averaged and cell size expressed as average cell diameter. The electrophysiological data were collected with the use of single-electrode continuous current clamp (AxoClamp-2B, Axon Instruments, Foster City, CA) and analyzed with Clampex 8 software (Axon Instruments).

After a stabilization period of 10 min, a large neuron was isolated. Criteria used for acceptable neurons are a resting membrane potential (V_m) of less than −45 mV and a peak action potential (AP) height greater than 0 mV regardless of the V_m, that is overshoot of the AP height over 0 mV. After a period of stabilization of the V_m of approximately 3 min, a current clamp protocol of increasing current injection into the cell was begun. During the absence of any external stimulus, any pattern of spontaneous activity (regular, bursting, or irregular) was noted. From each DRG, 2–5 cells were studied. To compare the sensitivity of neurons before and after paw incision in the different age animals, various membrane properties were measured as previously described.^23,24 Resting membrane potential (V_m) was first measured 3 min after a stable recording was obtained and was measured again after the end of the protocol. The current clamp protocol consisted of depolarizing currents of 0.05–4.0 nA (100-ms pulse duration) delivered in increments of 0.05 nA until an AP was evoked. The threshold current (rheobase) was defined as the minimum current required to evoke an AP. The AP and afterhyperpolarization (AHP) were determined from the trace generated by the rheobase depolarization. The AP voltage threshold was defined as the first point on the rising phase of the spike at which the change in voltage exceeded 50 mV/ms. The duration of the AP was measured at the AP threshold level. The AP amplitude was measured between the peak and the AP threshold. The input resistance (R_in) for each cell was obtained from the slope of a steady-state current-voltage plot in response to a series of hyperpolarizing currents of 100-ms duration, delivered in decreasing steps of 0.05 nA from 0.2 to −2 nA. The AHP amplitude was measured from the valley peak to the baseline; and the AHP duration was measured at amplitude half way between.

Statistics

Data were normally distributed and are presented as mean ± standard error. Withdrawal thresholds and electrophysiologic parameters were analyzed using analysis of variance between groups of similar ages for time and treatment and using Fisher’s protected least significant differences. Repeated measure was used for withdrawal thresholds. Between age differences were analyzed for baseline values only, except for rheobase, V_m, and R_in. Multiple comparisons were adjusted for using the Bonferroni correction where appropriate. Fisher’s exact test was used for differences between spontaneous oscillations between ages. All results were considered significant if the p-value was <0.05.

Results

Changes in Withdrawal Threshold

Withdrawal thresholds before surgery were lower in the one week old animals (5.7 ± 0.11 g; n=10) when compared to the four week old animals (46.2 ± 1.5 g; n=10; p<0.05) (fig. 1). After incision, the mechanical withdrawal threshold decreased significantly in both ages (p<0.05). The relative decrease for the 2 ages was not different with a percent decrease in threshold of 27% in the one week and 33% in the four week old rats at 6 hours after surgery. The threshold in the one week old animals increased at 24 hours and by 72 hours was no different from control threshold (100% of control). However, the threshold in the four week old animals at 24 and 72 hours remained below the control (48% and 48% decreased from control, respectively).

Figure 1.

Mechanical withdrawal thresholds in grams versus time in one week old (A) and four week old (B) rats. Baseline withdrawal thresholds are different (p<0.05). Thresholds decreased significantly after incision in both age animals (p<0.05). However, while thresholds remained significantly different from control in the four week old animals, thresholds were not different from control by 72 hours in the one week old animals. The (*) shows differences between control and surgery.

Spontaneous Activity of Neurons

None of the neurons in the study had spontaneous activity during the observation period. Representative tracings of the AP in neurons from one and four week old animals before surgery are shown in figure 2A. Five of 68 neurons from the one-week-old animals displayed spontaneous oscillations of V_m and multiple APs generated at threshold, reflecting increased excitability in these neurons (7.4% (C.I. 1.3, 13.5)) (fig. 2B). One of these cells was in the no surgery control group and there were 2 cells in each of the 24 and 72 hour post-incision groups. None of the 68 neurons from four week old animals demonstrated spontaneous oscillations (0% (C.I. 0, 4.4). The difference between the young and the old is not significant. However, the confidence intervals are provided to provide more information. Since this is a rare finding and the study was not designed to evaluate this finding, it may be an important difference.

Figure 2.

Action potential activation during current clamp step protocol. Representative action potential elicited in one and four week old animals are shown in figure 2A. Note the much greater change in membrane potential deflection from a fixed current in the one week old when compared to four week old animals at baseline. This is consistent with the greater resistance in the younger animal. Figure 2B demonstrates oscillations and multiple spikes in a cell from a one week old. This was not seen in cells form the older animals.

Differences in Large DRG neurons at baseline in the different ages

The V_m, rheobase and R_in all differed at baseline between the one and four week old animals (p<0.05) (fig. 3). V_m at baseline was less negative (more depolarized) in the one week old (−60.5 + 1.3 mV) when compared to four week old animals (−64.5 +1 mV) (p<0.05) (fig. 3A). Rheobase of the one week old is less (0.9 + 0.1 nA) when compared to the four week old prior to surgery (1.16 + .14 nA) (p<0.05) (fig. 3B). R_in was greater in the one week old animals before surgery (43.7 + 1.8 MΩ) than in the four week old animals (19 + 1.5 MΩ) (p<0.05) (fig. 3C). Large neuron size was larger in the four-week-old animals when compared to the cells from the one week old animals (p<0.05) (table 1). Overall differences between groups exist for AP threshold, AP amplitude, and afterhyperpolarization (AHP) duration (p<0.05) (table 1).

Figure 3.

A. Incision induced changes in Resting Membrane Potential (V_m), B. Rheobase (threshold current), and C. Input Resistance (R_in) during Development. In Figure 3A V_m is less negative in the one week old when compared to the four week old at baseline (p<0.05). Incision produced a significant change in V_m in both ages (p<0.05). Rheobase was lower in the one week old animals at baseline compared to the four week old animals in figure 3B (p<0.05). Incision produced changes in the Rheobase for both age animals (p<0.05). In figure 3C, R_in was higher in the one week old at baseline when compared to the four week old (p<0.05). Incision increased the R_in in both ages suggesting increased excitability in the neurons after the incision. The (*) shows differences between the one and four week old animals, while the (+) denotes difference from baseline.

Table 1.

Electrophysiologic characteristics of large cells from L5 DRG following incision at 1 and 4 weeks of age

Age	N	AP Threshold (mV)	AP Amplitude (mV)	AP Duration (ms)	AHP Amplitude (mV)	AHP Duration (ms)	Cell Diameter (μM)
1 Week
Control	22	−34.5 ± 3.4*	57.4 ± 2.0*‡	0.9 ± 0.1*‡	8.2 ± 0.9*‡	3.0 ± 0.2*‡	49.4 ± 0.7*
24 h	26	−40.0 ± 2.1†	66.1 ± 1.9†	2.2 ± 0.2†	12.1 ± 0.9†	3.1 ± 0.1	50.8 ± 1.4
72 h	20	−33.4 ± 3.0	50.9 ± 1.7	2.8 ± 0.2†	9.0 ± 0.7	4.5 ± 0.3†	49.3 ± 0.9
4 Week
Control	24	−43.2 ± 1.5‡	65.1 ± 2.2	1.7 ± 0.1	13.2 ± 1.4‡	2.1 ± 0.1	54.4 ± 0.6
24 h	20	−48.9 ± 1.1†	59.2 ± 1.4	1.8 ± 0.1	8.7 ± 0.9†	2.6 ± 0.2	51.8 ± 1.5
72 h	24	−43.5 ± 1.4	60.8 ± 2.2	1.7 ± 0.1	11.1 ± 0.8	2.8 ± 0.2	56.1 ± 1.8

All values are means ± SEM. The n is the total number of cells recorded from 6 animals at every treatment or time for each age. AP, action potential; AHP, afterhyperpolarization; DRG, dorsal root ganglia; L5, fifth lumbar vertebra; mV, millivolts; ms, milliseconds; μM, microns

^*denotes different between ages;

^†difference at baseline; and

^‡signifies different within age after incision.

Differences in large DRG neurons after incision at different ages

Incision produced a decrease in rheobase, a less negative (more depolarized) V_m, and an increase in R_in at both ages (p<0.05) (fig. 3). After incision, the V_m became less negative in both ages (p<0.05). In the four week old animal the V_m decreased at 24 hours when compared to baseline, but by 72 hours after the incision, the V_m was the same as at 24 hours. However, in the one week old animals V_m continued to become even less negative (more depolarized) at 72 hours. Rheobase decreased after incision at both ages (p<0.05). However, rheobase was similar between the one week and four week old animals at 24 hours, while at 72 hours, the largest difference was found between ages. Incision produced an increased in R_in in DRG neurons of both ages. However, the effect on R_in was greatest at 72 hours in the 4 week old animals, while in the one week old animals, the R_in was greatest at 24 hours and remained at the same level at 72 hours. Cell size was not different within each age (table 1). For the one week old animals, incision altered AP amplitude, AP duration, AHP amplitude, and AHP duration, but did not produce any significant change in AP threshold (table 1). For four week old animals incision produced significant effects only on AHP amplitude and the AP threshold.

Discussion

Differences in response to pain from various insults occur as a function of age and stage of development.^2,25–27 Understanding the etiology of these differences may help define changes which can either accentuate the pain signaling from the periphery or reduce it.²⁸ Noxious input from the periphery may provide greater input to the immature spinal cord and may result in long term alterations of neural processing after transient acute painful stimuli. The data presented in this study demonstrate differences between one and four week old animals in the electrophysiologic characteristics of large neurons from isolated DRG. Specifically, there is a less negative (more depolarized) V_m in the younger animals. This, in conjunction with the smaller rheobase, or threshold current needed to cause activation of the nerve, may result in greater excitability. In addition, incision in the hindpaw in the dermatome of neurons in the study results in changes in neuronal characteristics in all ages suggesting peripheral neuronal activity changes may in part drive changes in the spinal cord and elsewhere in the central nervous system in response to incision. Of possibly greater importance, the electrophysiologic changes that occur persist in the younger age group despite the withdrawal threshold returning to normal. This is a significant divergence between the ages. This suggests that changes are occurring in the young to mitigate the acute behavioral findings of altered thresholds. However, persistent input into the central nervous system may result in continued changes in processing of afferent stimuli which is masked by behavioral assessment. Thus, the opportunity to alter the input is ignored and may result in long-term changes in the spinal cord or even the brain leading to more subtle behavioral alterations and responses.¹³

Previous reports of evoked firing properties of the dorsal horn cells of the spinal cord in rat suggest that there is no significant difference in firing frequency, rheobase, adaptation, or regularity of action potential discharge at different ages after birth up until the third postnatal week.²⁹ However, this does not address differences in neuronal characteristics in the peripheral neurons. Peripheral neuronal input may be critical to normal development at the level of the spinal cord. It may also be responsible for altered development if input is out of proportion to that required for establishing and reinforcing normal connections. The synaptic reorganization and strengthening that occurs over the course of development may be related to the maturation of intrinsic neuronal characteristics.¹⁶ Reorganization occurs postnatally in the dorsal horn with A beta fibers being in the Lamina I and II initially and withdrawing over time.¹⁷ Work in this area suggests the changes that occur postnatally may be an activity dependent process.³⁰ This potential enhanced activity in the peripheral neuron from the lower V_m and the lower rheobase in the peripheral neuron may allow the immature neurons to contribute to the maturational organization in the dorsal horn. However, increased firing beyond this may be detrimental by providing an inordinate amount of signaling into the dorsal horn setting up circuits which may alter sensation and sensory processing remotely from the time of the surgery or tissue trauma. These same changes in the older animal, if they become established such as in nerve injury, whereby there is ongoing activity from even innocuous stimulation as a result of reduced thresholds, may alter circuits in the central nervous system and lead to chronic pain.

In this study, the DRG neurons were randomly selected and few of these actually innervate the location of the incision (previous unpublished results). Yet, a difference after incision is clearly demonstrated. This suggests changes in neurons from the injury may alter nearby neurons in the DRG not innervating the area of injury, since changes found in this study are too profound to attribute to one or two neurons in the DRG.¹⁶ The large neurons in the DRG likely represent the large myelinated A beta fibers based on conduction velocity characterization in a previous study.¹⁵ Von Frey filaments test in large part A beta fibers and the electrophysiologic data closely correlate with the mechanical withdrawal thresholds reported here for the older animals. The divergence of the mechanical thresholds and the electrophysiologic changes in the younger animals suggests the input to the spinal cord is still present in the young, but the responses are changed. This may be from where the large neurons synapse in the young spinal cord, modulation of the connections through descending pathways, or more rapid adaptation to the activity.¹⁷

Sensory hyperinnervation can occur in the periphery in the young which is more intense than that seen in response to the same injury later in life.^7,31 These sprouts are still coming into the same soma of the afferent neuron into the DRG and may alter sensitivity in the area of the wound through ongoing increases in signaling into the cell body and subsequent alterations in channels, receptors or other related mediators of the neuron responses. While A and C fibers are both involved in this localized, peripheral sprouting in the skin, it is the enhanced sprouting of the A fibers, and in particular the large A fibers in this preparation which are of greatest interest in the younger animal. In our preparation, these large neurons have characteristics which may render them more excitable. With input of these A fibers into more superficial lamina in the young, input from these fibers may be activating dorsal horn structures normally reserved for noxious signaling.¹⁷

The underlying differences inside the neurons responsible for the phenotypical differences in electrophysiological characteristics in our studies are not known. While these are still in vitro changes and the exact implications are not entirely clear, especially during current clamp depolarization, new evidence suggests that the cellular characteristics from in vitro preparations agree with the more natural states for most neuronal classes.³² Changes in calcium and sodium ionic currents have been found during development.^33,34 More specifically, the neurons from the younger animals may be more likely to have different types of inward currents from both calcium and sodium and as maturation occurs, greater numbers of neurons with fewer inward currents exist. In addition, in the younger cells, low threshold calcium currents are in greater abundance with fewer high threshold calcium currents.³⁵ This may explain the increased likelihood of multiple action potentials and oscillations in the younger neurons. Another possibility is the differential expression of ion channel subtypes as a function of development and even in response to the injury, or even regulated by changes in neuronal activity from the injured area.^36,37 Differences in Na_V1.9 between one week and adult animals with respect to A fibers have been shown, since normally Na_V1.9 is not present in adult A fibers and is present at one week.³⁶ This is contrary to Na_V1.9 in C-fibers which seems not to change during development. Postnatal increases in other sodium channel subtypes or ratios of other channels have also been described which may influence excitability.^38,39 These and other changes in ion channels, ratios, and expression will be crucial to understanding the basis for our findings and their implications.

Limited changes in the A beta fiber electrophysiologic characteristics have been described following incision in adult animals, but these changes were only assessed within the first 45 minutes after incision.⁴⁰ A delta fibers were sensitized in this study, but only A delta and C fibers were assessed for sensitization further in time after incision.⁴¹ However, these were all adult animals and further studies during development will provide knowledge of the changes in neuronal activity from incision to understand the impact in the young.

The data presented here demonstrate differences in electrophysiologic phenotype of large neurons from DRG as a function of development. The effects of incision on altering the underlying electrophysiologic phenotype of the large neurons are also presented. Further studies using voltage clamp will be needed to establish the role different ion currents may play in these findings, both at baseline during development and in response to incision. Studies will also be needed to determine the developmental regulation of these ion currents through protein and gene expression during development and in response to surgical incision. Most importantly defining the mechanism of the divergence in the behavior and the neuronal characteristics after incision in the young and understanding the impact of ongoing neuronal input at both ages will be critical in understanding postoperative pain. Through further study, the impact development has on peripheral neuronal responses to incision will allow more developmentally appropriate interventions to reduce the impact of surgical incision in the young.