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From the *Department of Anesthesia and Perioperative Care, University of California, San Francisco; Department of Anesthesia, Stanford University School of Medicine; Department of Anesthesiology and Pain Medicine; and Section of Neurobiology, Physiology and Behavior, University of California, Davis, California.
Address correspondence and reprint requests to E. Carstens, Section of Neurobiology, Physiology and Behavior, University of California, Davis, 1 Shields Ave., Davis, CA 95616. Address e-mail to eecarstens{at}ucdavis.edu.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: We recorded responses of rat sacral dorsal horn neurons to 20-s trains of electrical tail stimulation at different frequencies (0.3–10 Hz) and intensities (0.8–5 x stimulus threshold) during delivery of 0.7 to 1.3 minimum alveolar anesthetic concentration isoflurane. Summed responses (area under the curve [AUC] windup), initial response, absolute windup (AUC minus 20 times the initial response), and slope of windup were quantified.
RESULTS: Increases in stimulus intensity and frequency progressively increased AUC windup (P < 0.01 for both) and correlated with isoflurane concentrations required for immobility (R2 = 0.98 and 0.97, respectively). Increasing the isoflurane concentration significantly suppressed each measure of windup elicited by low-intensity and low-frequency, but not high-intensity and high-frequency stimulus trains. The initial response magnitude significantly correlated with slope of windup across stimulus intensities and isoflurane concentrations. The NMDA receptor antagonist MK801 significantly reduced windup (to 53%; P < 0.05) at 1 Hz.
CONCLUSION: Windup of dorsal horn neurons at low stimulus intensities and frequencies increases isoflurane requirements for immobility via a NMDA receptor-dependent mechanism. At high stimulus intensities and frequencies, windup was resistant to isoflurane consistent with larger anesthetic requirements for immobility.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Although these behavioral studies imply the importance of temporal and spatial summation, the specific neurophysiologic mechanisms underlying these processes remain unclear. Here we investigated a potential mechanism, windup, which is the progressive increase in the response of nociceptive spinal dorsal horn neurons to repeated C-fiber strength electrical stimulation (4). We hypothesized that the output of such neurons feeds into central pattern generators for rhythmic (purposeful) movement, and that depression of windup may partly underlie the immobilizing action of anesthetics.
Most previous studies of spinal nociceptive processing, including windup, and its modulation by anesthetics have examined lumbar spinal dorsal horn neurons (5–15), whereas MAC studies usually use stimulation of the tail (1–3,16), which excites nociceptive neurons in the sacral spinal cord (17–20). Few electrophysiological studies have characterized sacral neurons (17–19,21–23) and windup, and anesthetic modulation of sacral neuronal activity has not been investigated. We therefore evaluated windup of sacral dorsal horn neurons as a possible mechanism underlying the contribution of temporal and spatial summation to isoflurane requirements for immobility (2). We hypothesized that sacral neuronal responses would increase as a function of stimulus intensity and frequency, and that responses to high-intensity and high-frequency stimulation would resist anesthetic depression commensurate with the effect of these stimulus variables on anesthetic requirements to depress movement (2). Because MK801 depresses isoflurane MAC (2), we further hypothesized that MK801 administration during isoflurane anesthesia would depress neuronal windup in parallel with its effect on MAC (2). The results provide new evidence supporting a role for spinal neuronal windup in the immobilizing action of isoflurane.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Groups
After characterization of up to five units per animal, the last cell characterized was assigned to one of the groups listed in Table 1. Groups 1–4 investigated windup. Units exhibiting windup to a 20 s pulse train at 1 Hz were selected at 0.9 MAC. The stimulus threshold (T) was determined as described previously (6). The mean threshold was 4.3 ± 2.2 V (sd), range was 1.9–7.8 V. Stimulus trains were delivered at constant frequency (1 Hz) and different intensities (Group 1, n = 10 units), constant intensity (2 x T) and different frequencies (Group 3, n = 7), or constant frequency and intensity but different isoflurane concentrations (Group 2, n = 11). Group 4 (n = 7) was investigated for the effect of IV injection of MK801 (Sigma-Aldrich, Inc., St. Louis, MO) on windup evoked at different frequencies using constant intensity (2 x T) and isoflurane concentration (0.9 MAC). MK801 was infused at 2.5 µg · kg–1 · min–1 (n = 5), and after 60 min, the responses were again studied at each of the frequencies. MAC was also determined in these rats, as well as in rats in the other groups, using the standard method (1). In two of the rats, MK801 was then infused at 10 µg · kg–1 · min–1 and the responses were again studied after 60 min.
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Groups 5–7 were similarly investigated for variations in stimulus intensity (Group 5, n = 9), frequency (Group 6, n = 8), and isoflurane concentration (Group 7, n = 10) for units exhibiting "flat" responses that did not windup.
For Group 8 (n = 7 rats), we used a previously established protocol (2) to determine MAC fractions required to ablate movement evoked at constant frequency (1 Hz) and different stimulus intensities (2.5–50 V). Group 9 was similarly used to determine MAC fractions to stimulation at constant intensity (5 and 50 V) and variable frequency. Data from Group 8 were combined with previously published data (2) for other frequencies.
Data Analysis and Statistics
Neurons were classified as windup, flat, or winddown according to whether the 1-Hz stimulation evoked an increasing (slope 
0.8 action potentials/ stimulus for first seven stimuli delivered at 2 x T, 0.9 MAC), constant (–0.8 < slope < +0.8), or a decreasing (slope > –0.8) number of action potentials. Responses to 1 Hz stimuli were divided into the following latency ranges: 0–20 ms (A–β), 20–100 ms (A–
), 100–400 ms (C-fiber), and 400–1000 ms (after discharge, AD), consistent with previous studies (6) for which the conduction distance from hindpaw to lumbar cord was comparable to the distance from tail to sacral recording site. Using the combined A–, C-fiber, and AD ranges (20–1000 ms) (6,10), responses to the 1-Hz stimulus trains were expressed as follows: (a) area under the curve (AUC; i.e., total number of action potentials summed across all 20 stimuli), (b) initial response, (c) absolute windup, and (d) slope (action potentials/stimulus for the first seven stimuli) (5,10). Absolute windup is AUC windup minus 20 times the initial response and is a frequently reported index of windup (6,10,27–29). AUC windup includes the initial response. Subtracting the initial response level from responses to successive stimuli removes "baseline" responses and leaves only the component of the response that augments across repetitions, referred to as absolute windup. The 3 and 10 Hz frequency trains produced overlapping windup responses, and AUCs (A–β responses subtracted) were calculated at 1-s intervals for comparison with responses at 1 and 0.33 Hz.
Single, two factor, and repeated measures (where appropriate) analysis of variance (ANOVA) were used for overall comparisons of stimulus intensity, stimulus frequency, and isoflurane concentration on initial responses, AUC windup, absolute windup, and slope, using commercially available software (SPSS 9.0, SPSS, Chicago IL; GB-Stat, Dynamic Microsystems Inc., Silver Spring, MD). t-Tests were used to compare results of neuronal depths, windup during steps from 0.9 to 1.3 MAC, and effects of MK801. Correlation coefficients were used to compare AUC windup responses to MAC fractions. The population MAC was calculated using logistic regression analysis (SPSS). The MAC of rats in Group 4 (MK801) and the MAC fractions of rats in Group 8 (behavioral) were calculated as the average of the concentrations just permitting and just preventing movement. All data related to action potentials (AUC response, initial response, absolute windup, and slope) are presented as mean ± sem. All other data are presented as mean ± sd.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The mean MAC was 1.3 ± 0.1 (mean ± sd, n = 26) vol%. The mean arterial blood pressure measured in 10 rats remained >70 torr throughout the recording procedures.
Group 1: Effect of Stimulus Intensity on Windup Neurons (Spatial Summation)
The example in Figure 1 shows a progressive increase in the number of action potentials across the first seven stimuli (windup). Responses to each stimulus increased with stimulus intensity (Fig. 1, left column). At 1.3 MAC (Fig. 1, right column), the C-fiber and AD responses at each stimulus intensity were markedly attenuated.
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At stimulus intensities >1 T, windup occurred in the C-fiber (100–400 ms) and AD ranges (Figs. 2C and D) but not in the A–β or A– ranges (Figs. 2A and B). The initial response in the A–, C-fiber, and AD ranges increased with increasing stimulus intensity at 0.9 MAC (Figs. 2B–D). The C-fiber and AD windup curves (Figs. 2C and D) progressively shifted upward, with increased slope, as stimulus intensity increased. The mean initial response, AUC windup, absolute windup, and slope all increased significantly with increasing stimulus intensity (Fig. 2E; repeated measures ANOVA, P < 0.01 for all). Absolute windup and slope reached plateaus above 3 x T (Fig. 2F).
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At subthreshold stimulus intensities (<1 T) there was, as expected, little or no initial C-fiber response; however, later stimuli in the train elicited responses (Fig. 2E, 
) indicating that the mechanism underlying windup is still operative at low levels of stimulus intensity.
There was a significant correlation between the mean initial response and each measure of windup across stimulus intensities (R2 = 0.98, 0.93, and 0.90, for AUC, absolute windup, and slope, respectively; P < 0.01). Thus, spatial recruitment of additional nociceptive afferents by increasing the stimulus intensity significantly enhanced initial responses and windup.
Group 2: Effect of Isoflurane on Windup Evoked by Different Intensities at 1 Hz
There was a progressive downward shift in the windup curves (2 x T, 1 Hz, n = 11), as isoflurane increased from 0.7 to 1.3 MAC (Fig. 3A), with significant depression of the initial response and AUC windup (P < 0.01 for both) and slope (P < 0.05) but not absolute windup (P = 0.11) (Fig. 3B). The progressive decrease in windup with increasing isoflurane (Fig. 3A) or decreasing stimulus intensity (Fig. 2E) was similar. This similarity is emphasized in Figure 3C, in which the slope of the curve representing different stimulus intensities (
) was not significantly different from that representing different isoflurane concentrations (•). Thus, increasing stimulus intensity (i.e., spatial summation) increases windup in a manner similar to that observed with decreasing isoflurane concentration.
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In 7 units, we tested the effect of increasing isoflurane concentration from 0.9 to 1.3 MAC on windup elicited by four different stimulus intensities at 1 Hz. At the lower stimulus intensities (1.6 and 2 x T), each measure of windup was equally suppressed. At higher stimulus intensities (3 and 4–5 x T), the slope and absolute windup were not decreased and AUC windup was significantly less attenuated at the higher isoflurane concentration (Fig. 3D). Thus, windup responses elicited by stronger noxious stimuli are more resistant to anesthetic depression, similar to the increased anesthetic requirement for prevention of movement in response to more intense stimuli (2). That is, the anesthetic requirements to attenuate windup, and to prevent movement, are increased by spatial summation.
Group 3: Frequency-Dependence of Windup (Temporal Summation) and Effect of Anesthetic Concentration
At 0.9 MAC, windup increased with stimulus frequency from 0.33 to 3 Hz, with nearly continuous firing at 10 Hz. Figure 4A shows a progressive upward shift in mean windup curves and increased windup slope with increasing frequency. Responses to 3 and 10 Hz trains were quantified at 1-s intervals to allow direct comparison with lower frequencies (0.33 and 1 Hz), and are therefore higher due to summation of multiple responses evoked during the 1-s interval. At 0.33 Hz, there was a progressive increase in firing rate in the interstimulus interval presumably because of cumulative AD activity (Fig. 4A, 
). At 3 Hz, the initial 1 s response was approximately three times more than at 1 Hz (Fig. 4A, •), and subsequent responses quickly increased to a plateau similar to 10 Hz. At 10 Hz, responses reached a plateau at 5.5 ± 4.2 (sd) s., and the initial response was only about five times more compared with 1 Hz stimulation (Fig. 4A, 
). These results indicate that at 0.9 MAC, windup saturates quickly at higher stimulus frequencies.
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At 1.1 and 1.3 MAC isoflurane, initial responses, slope and AUC windup were dose-dependently reduced for stimulus frequencies below 10 Hz (Figs. 4B and C). Overall, there was a frequency-dependent increase in AUC windup at all isoflurane concentrations (Fig. 4D, P < 0.01). Increasing isoflurane from 0.9 to 1.3 MAC resulted in a significant, concentration-dependent suppression of AUC windup at 0.33 and 1 Hz (P < 0.05 for both), but not at 3 or 10 Hz (P = 0.11 and 0.61, respectively) (Fig. 4D).
Thus, temporal summation contributes to windup, and influences anesthetic requirements to suppress it. Isoflurane requirements to depress nociceptive neuronal responses, as well as movement (2), are larger at higher frequencies of noxious stimulation.
Group 4: Effect of MK801 on Windup Neurons
MK801 (2.5 µg · kg–1 · min–1 over 60 min) reduced the initial response and AUC windup at 1 and 10 Hz (Fig. 5A). AUC windup elicited by 1 Hz trains was significantly suppressed to 53% ± 12% (sem) of control (Fig. 5B, P < 0.05) and the initial response was suppressed to 64% ± 15% (P = 0.055). MK801 did not significantly affect AUC windup at 3 Hz, but suppressed it at 0.33 and 10 Hz (to 59% ± 12% and 79% ± 9%, respectively, P < 0.05). MK801 at a higher infusion rate (10 µg · kg–1 · min–1) further suppressed AUC windup at 0.33 and 1 Hz in 2 units. MK801 increased the spontaneous firing rate of two neurons.
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Isoflurane MAC during MK801 infusion (2.5 µg · kg–1 · min–1) was 0.8 ± 0.3 vol % (n = 5), and was significantly lower than the population MAC of 1.3% (P < 0.05).
Group 5: Effect of Stimulus Intensity on Flat Neurons
Nine flat responding neurons exhibited a stimulus intensity-dependent increase in the initial response that resulted in progressive parallel upward shifts in the windup curves. The effect of stimulus intensity on the initial response was not significantly different from that for windup neurons.
Group 6: Effect of Stimulus Frequency on Flat Neurons
Eight flat units tested with different stimulus frequencies exhibited relatively stable responses across trials that increased with frequency (0.9 MAC, 2 x T). Two neurons that had flat responses to 1 Hz trains produced windup to 3 Hz trains.
Group 7: Effect of Isoflurane Concentration on Flat Neurons
Isoflurane significantly suppressed the initial response and AUC (P < 0.01 for both, n = 10) in a concentration-dependent manner (1 Hz trains; 2 x T). Isoflurane suppression of the initial response of flat and windup responding neurons over the 0.7 to 1.3 MAC range was not different (NS, two-factor ANOVA).
Group 8: Effect of Stimulus Intensity (Spatial Summation) on MAC
The MAC fractions needed to ablate movement to 1 Hz electrical stimulation at various stimulus intensities are shown in Figure 6A. There was a significant (P < 0.01) linear correlation between mean MAC fraction and stimulus intensity with R2 = 0.98. Superimposed is a plot of mean AUC windup versus stimulus intensity (Group 1), which was also significant (P < 0.01) with R2 = 0.98. This comparison emphasizes the similar influence of spatial summation to increase both windup and the anesthetic requirements to prevent movement.
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Group 9: Effect of Stimulus Frequency (Temporal Summation) on MAC
The MAC fractions needed to ablate movement to constant (5 or 50 V) electrical stimulation at various stimulus frequencies are shown in Figure 6B. Superimposed is a plot of AUC windup versus stimulus frequency (Group 3). All linear correlations were significant (P < 0.01; R2 = 0.97, 0.99 and 0.99, for MAC fractions at 5 V, 50 V and AUC windup). This comparison shows the parallel effects of temporal summation to increase both windup and anesthetic requirements to prevent movement.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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latency response that did not windup (Fig. 2B). The initial response and AUC windup of sacral neurons progressively decreased across the 0.7–1.3 MAC range of isoflurane. In lumbar neurons, the initial response and AUC windup were similarly depressed in the 0 to 0.8 MAC range, but in contrast to the sacral neurons, AUC windup was unchanged or enhanced at 1.2 MAC isoflurane despite further depression of the initial response (6,8,10). We do not have a satisfactory explanation for this difference between sacral and lumbar neurons. Lumbar neurons exhibited a concentration-dependent reduction in initial response and AUC windup for the step from 0.8 to 1.2 MAC for halothane (6,10), similar to the effect of isoflurane for sacral neurons.
Windup in Relation to the Influence of Spatial and Temporal Summation of Noxious Stimuli on Anesthetic Requirements
There was a progressive increase in the initial response, AUC, and absolute windup with stimulus intensity (Fig. 2), consistent with spatial summation by recruitment of additional C-fibers. The linear relationship between stimulus intensity and AUC windup paralleled the effect of stimulus intensity on the MAC fraction required for immobility (Fig. 6A). This is consistent with the possibility that anesthetic modulation of windup in nociceptive neurons contributes to the influence of spatial summation on anesthetic requirements. The highest stimulus intensities elicited maximal windup (Fig. 2E) and thus might be considered "supramaximal." Windup elicited at the highest stimulus intensities was more resistant to anesthetic depression (Fig. 3D), consistent with behavioral observations that isoflurane requirements approach 1 MAC at the highest stimulus intensity (Fig. 6A), and that MAC is not affected by the application of one versus two supramaximal stimuli (1). Thus, anesthetic requirements are influenced by spatial summation of submaximal nociceptive inputs but approach 1 MAC as spatial summation saturates at a maximal or supramaximal level.
Windup also increased with stimulus frequency. The mean maximum response (approximately 60 action potentials/s) elicited by high intensity or high frequency trains (Figs. 2E and 4A) was similar to response rates of lumbar dorsal horn neurons to continuous 55°C skin heating or electrical hindpaw stimulation at 1 Hz (8,26). At higher isoflurane concentrations, windup elicited by 0.33 and 1 Hz stimuli was depressed, consistent with human studies reporting that isoflurane (30) and ketamine (31) reduced temporal summation (windup) of nociceptive flexion reflexes. The frequency-dependence of AUC windup of sacral neurons paralleled that of the MAC fraction for behavioral immobility (Fig. 6B), consistent with the idea that anesthetic depression of windup in spinal neurons contributes to the influence of temporal summation on anesthetic requirements. Windup elicited by higher-frequency (3 and 10 Hz) stimulus trains was reduced albeit to a lesser degree (Figs. 4B–D). This is consistent with the behavioral data showing that with a more intense stimulus, higher MAC fractions are required for immobility across all stimulus frequencies (Fig. 6B, ). To conclude, both spatial and temporal summation of nociceptive spinal neuronal responses appear to contribute to the anesthetic requirement for immobility. The stimulus- and frequency-dependence of windup and its depression by isoflurane correlated well with the influence of stimulus intensity and frequency on anesthetic requirements to prevent purposeful movement (Figs. 6A and B). These findings are consistent with the notion that dorsal horn neurons drive the generation of rhythmic movement responses that define the end point of immobility, and that windup of these neurons may be a mechanism underlying temporal summation involved in MAC (2).
Responses of flat-responding neurons also increased with stimulus intensity and frequency and were depressed by isoflurane in a concentration-dependent manner. Despite their inability to windup, such neurons provide a substantial nociceptive input to the spinal cord that contributes to anesthetic requirements along with depression of windup.
Initial Response as a Predictor of Windup
The magnitude of windup correlated with that of the initial response (Fig. 3C). Windup reflects cumulative depolarization of spinal neurons and is accelerated by DC depolarization of the neuron in slices (12). In our in vivo recordings, the initial response establishes a level of membrane depolarization, which may affect the rate of cumulative depolarization of the neuron by subsequent inputs. However, the slope of lumbar neuronal windup can be changed without affecting the initial response (Fig. 5A, and •), and conversely, the windup curve can be shifted downward with reduced initial response but little change in slope (6). This suggests that independent processes may govern the initial response (e.g., modulation of presynaptic input and/or postsynaptic membrane potential) and slope of windup (e.g., cumulative depolarization via NMDA and/or NK-1 receptor-mediated mechanisms). In the present study, isoflurane appeared to modulate both in parallel.
MK801
The reduction of sacral neuronal windup by MK801 is consistent with previous studies of lumbar neurons (32). At higher stimulus frequencies, MK801 reduced windup less compared its MAC-sparing effect (Fig. 6C), suggesting that the latter involves mechanisms other than suppression of dorsal horn neuronal windup. One possibility is that MK801 acts more at ventral horn neurons (e.g., central pattern generator, premotor interneurons, or motoneurons). Nitrous oxide, which blocks the NMDA receptor, more strongly depressed ventral than dorsal horn neurons (33). Ketamine, another NMDA blocker, also more strongly depressed noxious stimulation-evoked responses of motoneurons compared with dorsal horn neurons (34). In the clinical setting, progressive surgical trauma or repetitive maneuvers may contribute to windup of nociceptive neurons, thereby increasing anesthetic requirements, particularly when anesthetic concentrations are low.
In summary, sacral dorsal horn windup increased with increasing intensity and frequency of electrical stimulation of the tail. At low stimulus intensities or frequencies, windup was dose-dependently reduced by isoflurane, whereas at the highest intensities and frequencies, windup was resistant to anesthetic depression. These findings correlate well with isoflurane concentrations required to prevent movement, and support a role for dorsal horn windup in the isoflurane requirement for immobility. At high stimulus frequencies, MK801 reduced windup less compared with its MAC-sparing effect (2), suggesting that the sacral dorsal horn is not the only site mediating the influence of spatial and temporal summation on anesthetic immobility.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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; data from Dutton et al., 2003; R2 = 0.99). Superimposed is a plot of AUC windup (0.9 MAC, 2 x T) versus stimulus frequency (•; error bars: sem). Percent reduction from control at each frequency was calculated for MAC fractions and AUC windup, the corresponding values paired at each frequency, and correlation calculated. Inverse R2 = 0.60 (NS).
