I. Spinal recordings suggest that wide-dynamic-range neurons mediate sympathetically maintained pain

289

Puifz, 34 (I9SS) 289-x14 Bfsevier

Basic Section I. Spinal recordings suggest that wide-dynamic-range mediate sympathetically maintained pain

neurons

William J. Roberts and Mark E. Faglesong ~~ura~a~j~a~ Sciemes fnstiiute, Caad Samaritan Hospital and Medical Cents, (Received

6 January

1988, revision received 7 April 1988, accepted

Portlund, OR 97209 (U.S. A.) I1 April 1988)

In order to determine which classes of spinal neurons are capable of mediating s~~athetica~ly sumlnaFy and eoncIusions maintained pain, recordings were made from single somatosensory neurons in spinal cards of anesthetized cats. Each neuron was functionally identified with mechanical stimuli, and its responses to electrical stimulation of the sympathetic trunk were recorded. Nearly half (45%) of the wide-dynamic-range (WDR) neurons tested were activated by sympathetic stimulation, but none of the high threshold (n~iceptor-specifics neurons and ouly 17% of the low threshold neurons were activated, Sympathetic activation was most common for WDR neurons that had the foliowing: receptive fields proximal to the toes, few thresholds for mechanical activation, and both rapidly and slowly adapting responses to pressure. The predominant WDR response to sympathetic stimulation was long latency (> 1 set) excitation. Sympathetic activation of WDR neurons was abolished by each of the foifowing procedures: subcutaneous injection of local anesthetic, cooling of the receptive field with ice, and intravenous injection of the alpha-adrenergic blocker, phentolamine. The axons of some sympathetically activated WDR were shown to project to higher centers. These results indicate that WDR neurons are the only spinal nociceptive neurons activated by sympathetic efferent activity in this preparation. Therefore, WDR neurons, rather than high threshold neurons, are most likely to mediate the spinal component of sympathetically maintained pain. These results provide supporting evidence for our previous hypothesis [30] that sympatheticalfy maintained pain is mediated by myehnated mechanoreceptors acting on sensitized WDR neurons. Our results also demonstrate that sympathetic activation of WDR neurons is mediated by an aIpha-adrene~c mechanism in the skin. Key wards:

Sympathetic;

Pain; Spinal neurons;

Adrenergic;

Mechanoreceptors

The term sy~~~~~e~~c~~ ~i~t~~~~~ pain was recently introduced to functionally identify painful disorders such as causalgia and reflex sympathetic dystrophy, in which the pain is dependent on sympathetic efferent activity [30]. These disorders are characterized by a precipitating injury, spontaneous burning pain, a painful sensation during gentle mechanical stimulation (~l~d~a),

Coy~e~~~~~~~ to: Dr. William 3. Roberts, Ph.D., Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, 1120 N.W. 20th Ave., Portland, OR 97209, U.S.A. 5304-3959/88/$03.50

0 1988 Elsevier Science Publishers

and relief from the painful s~ptoms by sympathetic block [4,21,42]. Although these painful disorders have been recognized clinically for more than a century, the physioiogical mechanisms through which sympathetic efferent activity causes pain have not been determined. Hypotheses that have been advanced to explain these syndromes include: sympathetically induced ischemia and dystrophy [20]; s~path~tic activation of nociceptive afferents in the skin or in a nenroma [2,~0,13,19,36]; and sensitization of central pain pathways [20,21,42]. Each of these hypotheses expfains certain aspects of sympathetically maintained pain; however, none

B.V. (Biomedical

Division)

290

satisfactorily explains all or most of the clinical findings. Explanations for most clinical findings in these disorders were provided with our more recent hypothesis, which inco~orated new results from studies of sympathetic actions on primary afferent fibers in experimental animals 1301. We proposed that the essential dysfunction in these disorders is a persistent sensitization of spinal wide-dynamicrange (WDR) neurons, resulting in an abnormally high rate of firing in response to afferent input. The spontaneous burning pain associated with the syndrome is proposed to result from sympathetic activation of mechanoreceptors that project onto sensitized WDR neurons. A’flodynia is proposed to result from mechanical activation of the same of similar receptors that also project onto sensitized WDR neurons. This hypothesis differs from most previous hypotheses in proposing that mechanoreceptors, not nociceptors, mediate both the spontaneous burning pain and the allodynia that are characteristic of these disorders. Previous studies of sympathetic effects on cutaneous afferents led to the proposal that mechanoreceptors mediate the painful sensations in these disorders, These studies demonstrated sympathetic activation of several types of mechanoreceptors but not nociceptors, in the absence of tissue injury [ 1,27.32,33,35,40,44]. Other studies of spinal neurons showed that WDR neurons receive input from mechanoreceptors as well as nociceptors and that nociceptor input causes sensitization of these neurons; however, responses to sympathetic stimulation were not tested [9,2X,29,38]. The purpose of the present study was to test the spinal component of our hypothesis; specifically, we sought to determine which classes of spinal nociceptive neurons are activated by sympathetic stimulation and are therefore capable of mediating SMP. Results of this study are compared with chnical findings from humans.

Methods

Experiments were conducted of both sexes weighing 2.5-3.5

on 23 adult cats kg. Each animal

was anesthetized with either sodium pentobarbitai (35 “g/kg, i-p., n = 14) or ‘alpha-chloralose (60 mg/kg, i.p,, n = 9). Cannulae were placed in the right cephalic vein to administer anesthetic supplements and fluids (Ringer’s solution, 20 ml/h). in the right common carotid artery to measure arterial pressure, and in the bladder to collect urine. Inulin (MW = 5200, 53 mg/h) was continuously infused with the Ringer’s solution to increase the serum osmotic pressure, thus minimizing the spinal edema that otherwise develops after the dura is opened. Core temperature was maintained at 38°C. The experiment was terminated if the mean arterial pressure fell below 80 mm Hg. A bipolar stimulating electrode was pIaced on the right sympathetic trunk between L, and L, as illustrated in Fig. 1 and was insulated from surrounding tissues. A second electrode was placed on the left trunk at L,_ 5 in 5 animals to test for contralateral sympathetic effects. Each trunk was crushed rostra1 to the stimulating electrode to minimize input to spinal neurons from afferents projecting through the sympathetic trunk. The incision was closed with wound clips, and the animal was mounted in a spinal frame. Two la~nectomies were performed on each animal; the first to permit electrical stimulation of ascending fiber tracts in L,. and the second to

SKIN

Fig. 1. Schematic drawing of the experimental setup. The diagram shows the approximate positions af the stimulating electrodes in spinat segment I_2 and the recording pipette in L6. Also shown are the sympathetic stimulating electrode (SS), a sympathetic ganglion (SG), and a dorsal root ganglion (DRG) that contains both A- and C-afferents innervating the skin.

291

allow single unit recording from somatosensory neurons in L,_,. Ethanol (95%, 3 ml) was injected bilaterally into the paravertebral muscles (L,-L,) to irreversibly block contractions evoked by electrical stimulation in the spinal cord. This block allowed stable recording from spinal neurons without the use of systemic paralytic drugs. In the first 18 animals, the ipsilateral dorsolateral fasciculus was transected to block descending influences. However, this transection resulted in reflex vasospasm and a reduction in neuronal activity in nearby spinal segments; therefore, this procedure was discontinued. To identify somatosenso~ neurons having axons in ascending spinal tracts, the spinal cord was stimulated at L, through pairs of tungsten microelectrodes (1 mm tip exposure). The electrodes were placed manually in the left ventrolateral fasciculus and in the right dorsolaterai fasciculus. An additional pair of electrodes was placed in the right dorsal column in 9 animals. The exposed spinal cord was then covered with warm mineral oil.

Recording Low resistance (3-8 MJ2, 3 M NaCl) micropipettes were used to record extracellularly from single somatosensory neurons in spinal segments L 5-J. The amplified voltage from the pipette was filtered (0.1-10 kHz), and unitary spikes were converted to pulses with a window discriminator. Pulses from the window discriminator were counted by a rate monitor, which gave a voltage output proportional to the spike rate, averaged over the preceding second. This output voltage, together with the filtered signal and blood pressure, were photographed from a storage oscilloscope and stored on tape. As the recording electrode was advanced through the dorsal horn, the hind limb was stimulated manually to excite somatosensory neurons. A recorded unit was presumed to be a spinal neuron, not a primary afferent, if the spikes had a prominent negative component and if the stimulus-response characteristics and receptive field size of the unit differed markedly from those of known classes of primary afferents [6].

Unit classification

based on responses to mechanical

stimulation When a responsive somatosensory neuron was isolated in the recording, the cutaneous receptive field was determined with both non-noxious and noxious stimuli and was mapped onto a scale drawing of the cat hind limb. Non-noxious mechanical stimuli were delivered by Von Frey filaments, cotton swabs, blunt probes, and air puffs. Noxious mechanical stimuli were delivered by pinching small folds of skin with serrated forceps. Each neuron was classified according to the criteria described below. Low threshold high threshold or w~de-dynamicrange neurons. Each neuron was classified as low threshold, high threshold, or WDR, using conventional criteria [16]. Neurons maximally excited by non-noxious pressure were classified as low threshold neurons. Neurons consistently activated only by noxious pinch were classified as high threshold neurons, and those maximally excited only by noxious pinch but also responsive to non-noxious stimuli were classified as WDR neurons. Receptive field. Each neuron was assigned to 1 of 3 classifications based on the location and extent of its receptive field: (1) fields restricted to the toes; (2) fields excluding the toes; and (3) fields including both toes and more proximal skin. Adaptation rate. The rate of adaptation of each neuron to pressure was determined using nonnoxious manual stimulation of a 1 cm2 area in the center of the receptive field. The stimulus intensity was well above threshold but below damaging intensity. Units were assigned to 1 of 3 adaptation classes based on their responses to maintained non-noxious pressure: (1) rapidly adapting - respond for less than 2 set to sustained pressure; (2) slowly adapting - respond with little decrement in firing rate throughout a 5 set stimulus presentation; and (3) rapidly and slowly adapting - respond with a vigorous burst of activity at stimulus onset and with sustained activity at a lower rate throughout a 5 set stimulus. Threshold. The threshold for mechanical activation was determined by stimulating the center of the receptive field with each of 3 Von Frey filaments. Each neuron was classified as having a

292

threshold in one of the following ranges: g; (2) 1.2-3.5 g; and (3) > 3.5 g. Relative responsiveness to noxious and ous stimuli. WDR neurons were further to 1 of 2 classes: (1) neurons in which response to pinch was more than twice mal response to pressure; and (2) neurons the peak response to pinch was less than maximal response to pressure.

(1) < 1.2 non-noxiassigned the peak the maxiin which twice the

Unit classification based on responses to sympathetic stimulation Sympathetic effects on the activity of spinal neurons were tested with electrical stimulation of the sympathetic trunk for 30 set at 10 Hz with 0.1 msec pulses. Stimulus intensity was supramaximal for eliciting the following responses: piloerection on the tail; deflation of the small saphenous vein crossing the peroneal tendon; an increase in central arterial pressure due to hind limb vasoconstriction (typically lo-20 mm Hg); and an increase in skin conductance on the central pad of the ipsilateral hind paw. The experiment was terminated if sympathetic stimulation at 10 Hz failed to elicit these responses. Sympathetic excitation or inhibition. A neuron was classified as excited by sympathetic stimulation if it was previously inactive but fired more than 30 spikes over a 30 set period in each of 2 or more trials. Spontaneously active units were classified as sympathetically excited only if the rate of firing increased by at least 25% during sympathetic stimulation in each of 2 or more trials. Spontaneously active neurons were classified as inhibited if their firing rates decreased by at least 25% during the period of sympathetic stimulation in each of 2 or more trials. No effort was made to induce background activity in inactive neurons to test for inhibition. Sympathetic response latency. Responses to sympathetic stimulation were also classified as being of either long or short latency. Long latency responses began more than 1 set after the onset of sympathetic stimulation, whereas short latency responses began less than 100 msec after stimulus onset.

Tests of sympathetic effects on WDR neurons Sympathetic effects on some WDR neurons were tested at different rates of stimulation to determine whether the effects change qualitatively, as has been observed in other autonomic effector organs [23]. Stimulus rates of 2, 5, 10, 20. and 33 Hz were used, as these are within the range observed in human skin nerves during rest and various maneuvers [14]. The 5 different stimulus rates were presented for 30 set each in random order at 3 min intervals. In order to determine whether sympathetically evoked activity in WDR neurons was elicited through the actions of sympathetic efferent fibers on primary afferents in the periphery or by direct spinal projections from nerve fibers located in the sympathetic trunk, 3 sets of tests were conducted. In one, the alpha-adrenergic blocker phentolamine (Regitine, Ciba-Geigy) was injected intravenously (2 mg/kg) to block alpha-adrenergic actions in the periphery. In another, the local anesthetic lidocaine (1%) was injected subcutaneously into the receptive field to block conduction in peripheral efferent and/or afferent fibers. In the third test. ice was applied topically in the receptive field to depress neural function in the skin. With each test. responses in single neurons to sympathetic stimulation were recorded before and after these procedures. Localization of somas and axons The depth of the electrode tip from the cord surface was used to determine the approximate dorsoventral location of the soma for each neuron. These measurements were used to determine whether sympathetically activated neurons were localized in particular laminae within the dorsal horn and whether they were spatially segregated from non-activated neurons. Mediolateral locations were not determined. The axonal projections of most neurons were investigated by electrically stimulating each of the 3 ascending fiber tracts previously described and examining any responses recorded from the soma to determine whether the neuron was antidromitally activated.

293

Results

of Fig. 2B to illustrate the different responses to non-noxious pressure, noxious pinch and stimulation of the ipsilateral sympathetic trunk. These results demonstrate that WDR neurons are the only spinal somatosensory neurons that are both nociceptive and responsive to sympathetic stimulation. Only neurons that are both nociceptive and sympathetically activated are likely to mediate sympathetically maintained pain; therefore, the remainder of the results to be presented are exclusively from WDR neurons.

The results of this study are based on single unit recordings from 301 somatosensory neurons with cutaneous receptive fields on the right hind limb. No differences related to sex or type of anesthetic were observed in the responses of these neurons; therefore, data from all animals have been pooled. Sympathetic activation of cutaneous somatosensory neurons Responses to sympathetic stimulation were very different among the 3 classes of somatosensory neurons tested, as illustrated in Fig. 2A. Sympathetic stimulation activated nearly half of the WDR neurons sampled (57 of 128, 45%). However, sympathetic stimulation failed to activate any of 17 high threshold neurons and activated only 26 of 156 low threshold neurons (17%). Another difference in the responses between classes of neurons was that the magnitude of the sympathetically evoked response tended to be smaller in low threshold neurons than in WDR neurons, although this was not tested systematically. Representative responses from one unit in each class are shown in the peristimulus histogram

A@ 0

SYMPATHETICALLY NOT

ACTIVATED

Sympathetic activation in subsets of WDR neurons Many WDR neurons were sympathetically activated as shown above, but more than one-half were not. We therefore examined the incidence of sympathetic activation in subsets of neurons to determine whether specific stimulus-response characteristics were related to sympathetic activation. All neurons were segregated into subsets based on receptive field location, rate of adaptation to sustained mechanical pressure, threshold for mechanical activation, and relative responsiveness to non-noxious pressure versus noxious pinch. The incidence of sympathetic activation for subsets of WDR neurons is graphed in Fig. 3. (Thresholds for mechanical activation and relative

B

ACTIVATED

r

160

120

60

150

II,

HT

LT

WDR

-PRESS

PINCH

;o

SSi

Fig. 2. Graph of the incidence of sympathetic activation of somatosensory neurons (A) and (B) representative resporises in high threshold (HT), low threshold (LT), and wide-dynamic-range (WDR) neurons. The numbers of neurons that were (cross-hatched) and were not (open bars) activated by 10 Hz sympathetic stimulation are shown in A. The histograms in B illustrate responses of the 3 types of somatosensory neurons to non-noxious pressure (PRESS), noxious pinch (PINCH), and electrical stimulation of the ipsilateral

sympathetic

trunk (SSi).

A

RECEPftVE

FIELD

TOE

E3 I-

ADAPTATION

LOCATION

c

THRESHOLD

1

I

NONTOE

RATE

SA

SYMPATHETICALLY

c1.2g

TOE*NONTOE

D

RELATIVE

ACTIVATED

RESPONSE

PI>PR

RA-Sh

n

NOT

>3.5g

1.2g-3.sg

MAGNiTUDE

Pib>PR

ACTIVATED

Fig. 3. incidence of sympathetic activation in functional subsets of WDR neurons. Each graph shows the numbers of neurons that were (cross-hatched) and were not (open bars) activated by sympathetic stimulation at 10 Hz. The set of WDR neurons was subdivided according to the following criteria. A -~- receptive fieId location: fidds restricted to the toes (TOE). fields excfuding the toes (NONTOE), and fields including both toes and more proximal areas (TOE + NONTOE). B ^- rate of adaptation to sustained prew~re: rapidly adapting (RA), slowly adapting (SA). and both rapidly and slowly adapting (RA + SA). c’ -- threshold for mechanical activation. D .- relative magnitude of response tn pinch versus pressure: response to pinch slightly (PI z PR) or greatly (PI B PR) exceeded the response to pressure.

responsiveness were not determined in the early experiments; therefore, the total numbers of units listed in each of the following sections are not equal.) Rerepke fie& {Fig. 3A). None of 25 WDR neurons with receptive fields restricted to the toes were sympathetically activated. In contrast, 48 of 76 neurons (63%) with receptive fields excluding the toes were sympathetically activated, Of 27 units with receptive fields including both toes and more proximal areas, 50 (37%(yo)were activated. These differences are statistically significant (P ( (1.001, x3 - 31.2, crf= 2). Aduptation rate (Fig, 3B). Two of 24 WDR neurons (8%) that had only rapidly adapting responses to non-noxious pressure were activated by sympathetic stimulation. In contrast, 11 of 49 neurons (22%) classified as slowty adapting were activated by sympathetic stimulation, and 44 of 55 neurons (80%) classified as both rapidly and slowly

adapting were activated. These differences are statistically significant ( P < 0.001, x2 = 50.4, df‘== 2). Threshold (Fig. 3C). Of 73 WDR neurons having threshoids for mechanical activation less than 12 g. 50 (68%;) were activated by sympathetic stimulation. In contrast, only 3 of 15 (20%) units with thresholds between 1.2 g and 3.5 g, and 1 of 19 (6%) units with thresholds greater than 3.5 g, were sympathetically activated. These differences are statistically significant ( P < 0.001. x’ = 30.5. #= 2). Relufitie responsiue~exs to noxiaus and non-noxious stimuli (Fig. 3D). Sympathetic activation was observed in 23 of 43 WDR neurons (53%) in which the magnitude of the response to noxious pinch was more than twice that to non-noxious pressure, and it was observed in 29 of 51 units (57%) in which the response to noxious pinch was less than twice that to non-noxious pressure. These

295

differences are not statistically x* = 0.01, lif= 1).

significant

(P > 0.9,

Types of xympatheticaliy evoked responses in WDR neurons Four distinctly different types of responses to sympathetic stimulation were observed: long latency excitation, short latency excitation, long latency inhibition, and short latency inhibition. The predominant type of response - long latency was observed in 57 of 128 WDR excitation neurons (45%). This type of response was evoked by ipsilateraf sympathetic stimulation in 54 of the 57 neurons, by contralateral sympathetic slit&ation in 1 unit, and by both ipsilateral and contralateral sympathetic stimulation in 2 units. Responses representative of long latency excitation from 2 units are shown in Fig. 4A and B. Ail other types of responses were rare, as described below.

SYMPATHETIC

Short latency excitation was observed in response to ipsilateral sympathetic stimulation in only 3 of 128 WDR neurons (2@, and in response to contralateral stimulation in none. The short latency excitatory response of one unit is shown in Fig. 4C. Long latency inhibition was observed in only 1 WDR neuron (Fig. 4D). In that unit, inhibition of spontaneous activity developed slowly and persisted for several minutes beyond the 30 set of ipsilateral sympathetic stimulation. That unit was also inhibited by non-noxious pressure applied adjacent to the excitatory receptive field. Short latency inhibitory responses to sympathetic stimulation were observed in 3 of 128 WDR neurons (2%). Two of these units were inhibited by ipsilateral and one by contralateral sympathetic stimulation. Representative responses from one unit are shown at 2 different sweep speeds (Fig. 4E, F).

EXCITATION

SYMPATHETIC

INHIBITION

A

~~ -PRESS PINCH

PRESS

PINCH

-PRESS PiNCX

ssi

SSC

40-

6 ifi v)

20.

2 Y h _uJ

is se.2

SSi Fig. 4. Types of responses to sympathetic stimulation in WDR neurons. The units in A and B gave long Latency excitatory responses to stimulation of the ipsilateral (SSi) and contralateral (SSc) sympathetic trunks respectively. Short latency excitatary r’esponses to single sympathetic stimuli (arrows) are illustrated with two oscilloscope traces from one neuron in C. Long latency inhibition of another WDR neuron in response to SSi is shown in D. Short latency inhibition of spontaneous activity is illustrated in response to SSc of another unit in E. The spike trace in F was recorded from the same neuron as in E, but at a faster sweep speed showing both neuronal spikes and stimulus artifacts. The time scale shown in E applies aiso to A, B and D.

2 Hz

20

I I

I

Ph,vsiological basis for sympathetically evoked activit\, Three tests were conducted to determine whether sympathetic-sensory coupling in the skin was responsible for the long latency excitation evoked by sympathetic stimulation. These tests consisted of intravenous injection of phentolamine, subcutaneous injection of lidocaine, and topical application of ice in the receptive field. Intravenous injection of an alpha-adrenergic blocker, phentolamine, was used to test both for an alpha-adrenergic mechanism and a peripheral

A

,

I I

1

CONTROL

60

PRESS

I SSi

,

1

15 set

B

PHENTOLAMINE

c

RECOVERY

Fig. 5. Graded responses of one WDR neuron to stimulation of the sympathetic trunk at different rates (2--33 Hz).

Sympathetic activation of WDR neurons at different stimulus rates Twelve sympathetically activated WDR neurons were tested with 5 different rates of sympathetic stimulation, ranging from 2 to 33 Hz. Representative results from one are shown in Fig. 5. In all 12 neurons, the magnitude of the response was directly related to stimulus rate, and the type of response was the same at all rates of stimulation. In no unit was excitation observed at one stimulus rate and inhibition at another, or sequential excitation and inhibition at any one stimulus rate.

40

401

1

6 0

15 set Fig. 6. Effect of peripheral alpha adrenergic block on sympathetically evoked activity in a WDR neuron. The activity evoked by PRESS and SSi (10 Hz) is shown in A. intravenous administration of phentolamine (2 mg/kg) abolished the response to SSi as shown in recording B, made 6 min after injection. Partial recovery of the excitatory response 45 min later is shown in C.

297

site of sympathetic action, as phentolamine does not readily penetrate the blood-brain barrier. All 4 WDR neurons tested gave results similar to those shown for one unit in Fig. 6. The response of this unit to sympathetic stimulation prior to administration of the drug is shown in A. Subsequent injection of phentolamine (2 mg/kg) completely abolished the sympathetically evoked activity (Fig. 6B). Partial recovery of the response was observed 45 min later (Fig. 6C). All units remained responsive to mechanical stimulation of the skin throughout these tests (not shown). These results indicate that a peripheral alpha-adrenergic mechanism is responsible for sympathetically evoked activity in WDR neurons. To further establish that the sympathetic-sensory coupling occurs in the skin, single unit activity was recorded before and after injection of 1% lidocaine into the receptive field. Fig. 7A shows the activity evoked by non-noxious pressure and by sympathetic stimulation in one unit prior to the injection. Subsequent injection of lidocaine completely abolished sympathetically evoked activity, although the unit was still responsive to pressure,

A

CONTROL I

6C

.I,.

J

PRESS

B

SSi 60

LIDOCAINE

l&l PRESS

/ SSi

15 set

Fig. 7. Effect of subcutaneous lidocaine on sympathetically evoked activity in a WDR neuron. The histogram in A shows responses to both PRESS and 10 Hz SSi before 1% lidocaine was injected in the receptive field, and B shows responses to the same stimuli after lidocaine injection.

10

5

~I

I

6

0

1000

1

SYMPATHETICALLY

1

NOT

2000

CELL

ACTIVATED

ACTIVATED

DEPTH

3000

4000

(pm)

Fig. 8. Depths of WDR neurons tested. The numbers of sympathetically activated (filled bars) and non-activated (hatched bars) neurons are plotted as a function of the depths from the surface of the spinal cord at which they were recorded.

which activates myelinated mechanoreceptors (Fig. 7B). Additional evidence that sympathetic-sensory coupling in the skin is responsible for sympathetically evoked spinal activity was obtained by applying ice to the receptive fields of 3 WDR neurons. Cooling the skin reversibly blocked or profoundly depressed the excitatory responses of these neurons to sympathetic stimulation and reduced the magnitude of their responses to mechanical stimulation (not shown). The rapid cooling also evoked a transient discharge in these neurons prior to depression of the sympathetically evoked response. Soma depths and axonal projections Estimated soma depths of sympathetically activated and non-activated WDR neurons are graphed separately in Fig. 8. Both populations were widely distributed throughout the dorsal horn, but the mean depths of sympathetically activated and non-activated units were slightly different (mean + S.D. = 1639 pm + 575 and 1866 pm + 567, respectively). Electrical stimulation of ascending fiber tracts was used to investigate the axonal projections of WDR neurons. Axons of both sympathetically activated and non-activated neurons projected through the contralateral VLF, the ipsilateral DLF,

TABLE

I

AXONAL

PROJECTIONS

OF WDR NEURONS

This table shows the numbers (SS + ) and non-activated (SS -) antidromically to stimulation of fasciculus (VLF), the ipsilateral and the ipsilateral dorsal column

of sympathetically activated WDR neurons that responded the contralateral ventrolateral dorsolateral fasciculus (DLF). (DC).

-I

ss+ ss-

VLF

DLF

DC

6 15

11 7

1 I

and the ipsilateral DC, as shown in Table I. Although the number of sympathetically activated neurons with axons in the DLF was greatest, the differences in the numbers of activated versus non-activated units in the 3 tracts were not statistically significant.

Discussion Principal findings

This study shows that the incidence of sympathetic activation differs among neurons comprising the 3 classes of somatosensory neurons. Sympathetic stimulation activated 45% of the WDR neurons tested, only 17% of the low threshold neurons, and 0% of the high threshold neurons. Both the absence of sympathetic activation in high threshold neurons and the relatively high incidence of activation of WDR neurons are consistent with results from previous studies of sympathetic actions on cutaneous afferent fibers. Those studies showed that sympathetic activation does occur in certain types of low threshold mechanoreceptors but not in nociceptors, except under conditions that do not apply to the present study [1,27,31-35,401. It is therefore expected that sympathetic activation will not occur in high threshold neurons, which are activated only by noxious stimuli, but sympathetic activation is expected to occur in WDR neurons, which receive input from both nociceptors and low threshold mechanoreceptors.

The failure of high threshold neurons to respond to sympathetic stimulation and the relatively high incidence of activation of WDR neurons suggest that only WDR neurons are capable of mediating sympathetically maintained pain. as proposed earlier [30]. Implicit in this suggestion are the assumptions that high rates of firing in WDR neurons can elicit painful sensations [24] and that activity in low threshold neurons is not painful, since these neurons respond maximally to non-dama~ng stimuli [12,15,18,29]. Our finding that the incidence of sympathetic activation was much less in low threshold neurons than in WDR neurons was unexpected because both classes of neurons receive excitatory input from the same types of low threshold mechanoreceptors known to be sympathetically activated [45]. The reason for this difference in incidence is not known: however, 2 explanations seem plausible: (1) The lower incidence of sympathetic activation of low threshold neurons relative to WDR neurons may be due to segregation of the central terminals of mechanoreceptors, not all of which are sympathetically activated [33,34]. The central terminals of sympathetically activated mechanoreceptors may project onto WDR neurons. and sympathetically non-activated afferents may terminate on low threshold neurons. Such segregation of afferent terminals would result in the observed differences in the incidence of sympathetic activation; however, the present results are the only evidence suggesting such segregation. (2) The lower incidence of sympathetic activation of low threshold neurons may be due to greater inhibitory input onto low threshold neurons than WDR neurons from sympathetically activated mechanoreceptors. An ‘inhibitory surround’ input might be more advantageous in low threshold neurons, which presumably participate in the localization of tactile stimuli ]37], than in WDR neurons, which are more likely to participate in the detection of tissue damage. Mechanoreceptor-mediated inhibition has been observed in both low threshoId and WDR neurons [5,12,13,26,39]; however, the relative inhibitory input to the 2 classes of neurons from mechanoreceptors has not been studied systematically.

299

The remainder consideration of neurons and the clinical findings cally maintained

of the discussion is limited to sympathetic actions on WDR relations~p of these results to from patients with sympathetipain.

Sympathetic activation in subsets of WDR neurons The incidence of sympathetic activation was greatest in subsets of WDR neurons with the following stimulus-response characteristics: receptive fields that excluded the toes, both rapidly and slowly adapting responses to pressure, and low thresholds for mechanical activation. Sympathetic activation was found not to be related to the relative response magnitudes of WDR neurons to non-noxious and noxious mechanical stimuli. This subsection focuses on physiological mechanisms that may explain these associations between sympathetic activation of a neuron and its stimulus-response characteristics. Receptive field. Sympathetic stimulation activated 63% of the WDR neurons having receptive fields that excluded the toes, but activated none of the WDRs having receptive fields restricted to the toes. This difference cannot be explained by failure to activate sympathetic efferent fibers innervating the toes, as both vasomotor and sudomotor responses to sympathetic stimulation were verified on the toes of all animals. More likely, this discrepancy results from differences in sympathetic innervation of glabrous and hairy skin; for example, only in glabrous skin are vasodilatory fibers present and pilomotor fibers absent [17]. Unfortunately, sympathetic actions have not been tested on primary afferents innervating glabrous skin, so it is not known whether sympathetic effects on afferent activity differ in glabrous and hairy skin. Adaptation rate. WDR neurons having both a rapid and a slow component in their response to mechanical pressure in the receptive field were much more likely to be activated by sympathetic stimulation than were WDRs in which the response was only rapidly or slowly adapting (80% vs. 8% and 22% respectively). That few rapidly adapting neurons were sympathetically activated is easily explained. The principal rapidly adapting mechanoreceptor in hairy skin is the hair afferent, and most receptive

fields encountered in this study included only hairy skin. Because most hair afferents are desensitized, not activated, by sympathetic stimulation in cats [1,27,35], sympathetic efferent activity would not be expected to increase the excitatory input to rapidly adapting WDR neurons from hair afferents unless piloerection occurs in the receptive field [34]. It is more difficult to explain the relatively low incidence of sympathetic activation among WDR neurons having only slowly adapting responses to pressure, as many slowly adapting mechanoreceptor afferents are responsive to sympathetic stimulation [31,33,34]. One possible explanation is that slowly adapting WDR neurons are simply less responsive to mechanoreceptor input than are neurons with both rapidly and slowly adapting responses to pressure. This explanation is supported by our observation that 89% of the WDR neurons with both rapidly and slowly adapting responses had thresholds of less than 1.2 g. In contrast, only 42% of the neurons with only slowly adapting responses to pressure had thresholds of less than 1.2 g. These different thresholds probably result from differences in neuronal responsiveness rather than afferent sensitivities, as all low threshold mechanoreceptors have thresholds well below 1.2 g. More studies would be necessary to resolve this issue. Threshold. Sympathetic activation was most common in WDR neurons with relatively low mechanical thresholds (< 1.2 g). We attribute this finding to the fact that responses to both mechanical and sympathetic stimulation are mediated by low threshold mechanoreceptors; therefore, the neurons most responsive to mechanical stimulation would be expected to be most responsive to sympathetic stimulation, assuming that both mechanical and sympathetic stimuli act on the same mechanoreceptors. Relative response magnitude, The relative response magnitude of a neuron to non-noxious pressure and to noxious pinch was found not to be related to the incidence of sympathetic activation in WDR neurons. This absence of correlation’ between relative response magnitude and sympathetic activation indicates that sympathetic activation of WDR neurons is independent of the

relative efficacy of nociceptor input. This finding seems reasonable since sympathetic activation of WDR neurons appears to be mediated by low threshold mechanoreceptors. Combined subsets. The present results indicate that the incidence of sympathetic activation is related to receptive field location, rate of adaptation to pressure, and threshold for mechanical activation. The highest percentage of sympathetically activated neurons, based on any one of the above subsets, was 80% {rapidly and slowfy adapting neurons). However, the incidence of sympathetic activation was greater when a combination of certain characteristics was used to identify a population of neurons. For example, sympathetic activation occurred in 92% of the 39 WDK neurons that had all of the following characteristics: receptive fields that excluded the toes, rapidly and slowly adapting responses to pressure, and mechanieai thresholds less than 1.2 g. These test&s indicate that stimulus-response characteristics of WDR neurons can be used to reliably predict whether or not a unit wilf be sympathetically activated; such information may facilitate future studies of this population of neurons. Physiolagical bases for the 4 types of symputhetical!v evoked responses Most WDR neurons in this study had only one type of response of sympathetic stimulation long latency excitation. The physiological basis for this response was investigated with several procedures reviewed in the next paragraph. Other types of responses to sympathetic stimulation were rare in this study and will be discussed only briefly. Lvng latency excitation. The results of this study show that sympathetic-sensory coupling in the skin mediates the long latency excitatory response in WDR neurons. This coupling was demanstrated by 3 procedures: intravenous administration of phentolamine; subcutaneous injection of lidocaine; and topical application of ice. Phentolamine, an alpha-adrenergic blocker that acts peripherally when administered intravenously, abolished ~~~pathetica~~y evoked activity in WDR neurons. Additions direct evidence that the responses are mediated by sympathetic actions in the skin was obtained by blocking neural elements

in the skin with local anesthetic and ice. Both of these procedures blocked or attenuated sympathetic activation of WDR neurons. Collectively, these results establish that the long latency exoitatory responses to sympathetic stimulation are mediated by sympathetic-sensory coupling in the skin, as expected from studies of primary afferents 1322351, tong latency ~~hjb~~io~. Sympathetic stimulation produced long latency inhibition in one WDR neuron. Non-noxious pressure applied adjacent to the excitatory receptive field also inhibited spontaneous activity in this neuron. We therefore attribute the long latency inhibitory response ta sympathetic activation of mechanoreceptors with inhibitory input onto the WDR neuron. Inhibitory responses to sympathetic stimulation are probably more common than our results indicate because we did not induce the background discharge necessary for detection of inhibitory responses in extracellufar recordings. S’hort &ency excj~a~~on and ~nh~bj~~o~.The short latency responses to sympathetic stimulation, observed in 6 of 123 WDR neurons, differed greatly from long latency responses. The short latency responses began too early to be mediated by conduction in non-myelinated sympathetic efferent fibers projecting to receptive fields on the hind limb. Furthermore, short latency responses were evoked by single stimuli applied to the sympathetic trunk; single sympathetic stimuli never evoked activity in cutaneous afferent fibers in our earlier studies funpubhshed observation). These findings indicate that the short latency responses are not mediated by sympathetic-sensory coupling in the skin. It is more likely that the short latency responses result from electrical activation of visceral afferent fibers that are known to project through the sympathetic trunks and terminate on spinal somatosensory neurons [8,13]. S,vmpathetic effects at different srimulus rates Each WDR neuron showed qualitatively the same response to sympathetic stimulation at all rates of stimulation, atthough the magnitude and time course of the response changed with stimulus rate. There was no evidence of multiple effects at higher stimulus rates, as has been reported in

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studies of parasympathetic gland [23].

effects on the parotid

Soma depths and axonal projections The cell bodies of both sympathetically activated and non-activated WDR neurons were widely distributed throughout the dorsal horn, as estimated by the depths of the electrode tip from the cord surface at the point of entry. However, the mean depth of activated neurons was less than that of non-activated neurons (1639 pm and 1866 pm, respectively). Although this difference in mean depth suggests some degree of segregation between the two populations, we attribute this finding to mediolateral differences in the location of sympathetically activated and non-activated neurons. WDR neurons with more proximal receptive fields, which had a higher incidence of sympathetic activation than units innervating more distal areas, are located laterally in the dorsal horn [46]. The lateral regions of the dorsal horn are closer to the surface of the cord; consequently, the mean depth of neurons located laterally is less than that of units in the medial dorsal horn. Axons of sympathetically activated WDR neurons were found to project rostrally through 3 major ascending tracts: the contralateral ventrolateral fasciculus, the ipsilateral dorsolateral fasciculus, and the ipsilateral dorsal columns. Therefore, any or all of these pathways may be involved in mediating sympathetically maintained pain. Comparison of present results with clinical findings The present study was designed to improve our understanding of the physiological mechanisms responsible for sympathetically maintained pain in humans. The remaining discussion considers whether our results are consistent with clinical findings from patients and whether conclusions can be drawn regarding physiological mechanisms. This discussion will be conceptually guided by the hypothesis that sympathetically maintained pain is mediated by low threshold mechanoreceptors acting on sensitized WDR neurons [30]. Mechanoreceptors versus nociceptors. Our findings that WDR neurons are sympathetically

activated and that high threshold neurons are not activated suggest that sympathetic actions on low threshold mechanoreceptors, not nociceptors, are responsible for activation of the spinal neurons. Clinical interventions in humans similarly suggest that large diameter, low threshold mechanoreceptors mediate sympathetically maintained pain and allodynia. Selective pressure block of large myelinated afferents, with sparing of conduction in non-myelinated nociceptors and thermoreceptors, has been shown to abolish the spontaneous burning pain and allodynia in patients with these disorders [7,25, and J. Ochoa, pers. commun.]. Neurochemistry. The alpha-adrenergic blocker, phentolamine, was shown in this report to abolish sympathetically evoked activity in WDR neurons, indicating that the sympathetic-sensory coupling is dependent on an alpha-adrenergic mechanism. This finding is consistent with clinical reports that infusion of a noradrenergic depletor, guanethidine, into the painful area effectively abolishes the spontaneous burning pain and allodynia that are characteristic of sympathetically maintained pain [3,21,22]. Neuroma coupling. These experiments showed that spinal nociceptive neurons can be sympathetically activated in the absence of nerve injury. This result is consistent with clinical findings that sympathetically maintained pain occurs in people with or without nerve injury [21]. Both findings indicate that sympathetic sensory coupling in a neuroma is not necessary for sympathetic activation of spinal nociceptive neurons. Such coupling does occur in experimental neuromas [11,19,36,43], and it may contribute to sympathetically maintained pain that develops long after a nerve injury, but it is not a necessary condition. Sympathetic Sympathetically hyperactivity. maintained pains are sometimes thought to be associated with sympathetic hyperactivity, although many clinical investigators have cautioned that sympathetic hyperfunction is not consistently related to pain in these disorders [4,21,42]. Our finding that activity in WDR neurons can be evoked by electrical stimulation of the sympathetic trunk at rates as low as 5 Hz shows that high rates of sympathetic activity are not necessary to evoke firing in spinal nociceptive neurons.

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However, it is premature to extrapolate quantitatively from our results directly to painful conditions in humans because of a lack of knowledge about species differences, anesthetic effects, and rates of WDR activity that evoke painful sensation. Skin cooling. Our demonstration that cooling of the skin depresses sympathetic activation of WDR neurons suggests that the cool compresses often employed by individuals to reduce sympathetically maintained pain [42] are effective because the cooling depresses sympathetic-sensory coupling in the skin. Sympathetic responses from toes. Our finding that WDR neurons with receptive fields restricted to the toes are not activated by sympathetic stimulation is not consistent with reports that sympathetically maintained pain can involve the digits in humans [41,42]. This apparent discrepancy may be due to species differences and/or anesthetic effects or to sympathetic-sensory coupling in neuromas in humans, however, this issue cannot be resolved with present knowledge. Sympathetically maintained pain u sensory abnormality. In most humans, activation of low threshold mechanoreceptors through non-damaging pressure or sympathetic arousal does not result in painful sensation; however, in individuals with sympathetically maintained pain, both pressure and sympathetic efferent activity result in pain [4,20,21,41,42]. These differences are most easily attributed to differences in the excitability of spinal pain pathways [30]. Numerous studies have shown that some WDR neurons are sensitized by nociceptive input like that which is likely to occur during a precipitating injury [9,28,29,38]. Such sensitization is likely to result in exaggerated responses to both mechanically and sympathetically evoked activity in mechanoreceptors. If an abnormal sensitization persisted, it would explain the painful responses to mechanoreceptor input. Conclusions. Except for the absence of sympathetic activation of WDR neurons with receptive fields on the toes of cats, the findings of the present study are consistent with clinical findings in sympathetically maintained pain and with the hypothesis [30] that this pain is mediated by sympathetically activated mechanoreceptors and

sensitized WDR neurons. We are currently testing whether sensitization of WDR neurons enhances responses to sympathetic stimulation. It remains to be shown that a persistent state of sensitization of WDR neurons exists in individuals with this disorder.

Acknowledgements The authors wish to thank Drs. Paul Cordo and Ronald Kramis for critical readings of the manuscript. We are also grateful for financial support of this research provided by the USPHS (NS 13447) the Medical Research Foundation of Oregon, Good Samaritan Hospital, and BRSG Grant No. RR 05593 from the NIH.

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