Response of cat skin mechanothermal nociceptors to cold stimulation

Brain Research Bulletin,

0361~9230/85$3.00 + 40

VoI. IS, pp. 529432, 1985.e Ankho Intemation~ Inc. Printed in the U.S.A.

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Response of Cat Skin.Mechanothe~~ Nociceptors to Cold Stimulation JEAN-LOUIS

SAUMET,*’

SYLVIANE

CHERY-CROZEt

AND ROLAND

DUCLAUXS

*Laboratoire de Thermore’gulation et de Mktabolisme Energe’tique, C.N.R.S. UA 181 Universit& Claude Bernard, 69373 Lyon Cedex 8, France ?INSERM U 45, Hopital E. Herriot, Pavilion H Bis, 49374 Lyon Cedex 2, France ~Laboratoire de Neurophysio~ogie~ Centre ~ospital~er Lyon-Sud, 69310 Pierre Benite, France Received 6 March 1985 SAUMET, J.-L., S. CHERY-CROZE AND R. DUCLAUX. Response of cat skin mechanothermal nociceptors to cold BRAIN RES BULL E(5) 529532, 1985.-In the cat skin, the activity of 5 mechanothermal nociceptors has been studied to cold nociceptive stimuli. The frequency of discharge is very low (maximum of 2.0 Hz). The repetition of very cold stimulations decreased the discharge threshold of these receptors. After -5°C stimulus, the mechanothermal nociceptors were completely inhibited during 3 to 7 minutes. These mechanothermal nociceptors should have an important function in cold pain. stimulation.

Cold stimulation

Nociceptors

IN human beings different skin temperatures are needed in order to evoke cold pain according to the stimulus parameters. Dipping the entire hand in 18°C water during one minute induced a transient faint pain [14] whilst decreasing cutaneous temperature at a rate of 1.3”C set-* with a 6.5 cmZ Peltier device provokes a pricking pain only at about 10°C 181. With a pun&ate stimulator temperature must reach -40°C during 0.4 set to provoke a pure pricking pain sensation. Moreover, it was shown that the intensity of cold pain is linearly correlated with that of a cold stimulus between 2OYZand 0°C 151. In order to explain this pain sensation, it is necessary to know which cutaneous sensors are tiring at this low temperature. Such a study may be conducted in animals. Cutaneous nociceptors in monkeys have been shown to respond to cutaneous cooling from 30°C down to 10°C [12,13]. This group includes nociceptors with A S and C fibers. Freezing the skin may evoke a few impulses in some units of this type [4, 12, 13, 181. The number of impulses evoked by ice temperature would depend upon duration; LaMotte and Thalhammer [ZO]showed that stimulation for 20 set at an ice temperature evoked a mean of6.5 and 4 impulses for A S and C fiber nociceptors respectively. These receptors may therefore play a part in the perception of cold pain. This hypothesis is all the more probably as mechanothermal afferents represent 40 to over 90 percent of the total randomly sam-

Threshold

pled afferent C fibers in both cats and monkeys [2, 3, 181. Moreover, they respond to nociceptive stimuli of different types of energy: thermal and mechanical. Their responses to heat stimuli are well documented [l, 2, 9, 12, 131 but their intensity-response functions to cold stimuli have been quantifled in only 5 cells, of which 4 were C mechanical cold units and 1 was a C polymodal noclceptor [13]. For these cells, stimulus-~s~nse intensity relations were represented by power functions. This shows that these receptors may encode pain sensation, However only a small number of neurones has been studied at very low temperature. It would therefore seem necessary to carry out additional experiments in order to verify their participation in pain. METHOD

In this work, we have recorded the extracellular action potentials from cat mechanical nociceptors using one tungsten microelectrode of 0.9 Megohm pricked into a spinal ganglion between Tg and T,*. The receptive fields of mechanot~~~ nociceptive units were identified by the following criteria: absence of spontaneous activity; no activation to touch; brief discharge to prick, pin-prick and under radiant heat brief incursion around 50°C. After identification, the mechanical threshold was measured with calibrated Von Frey hairs. The response to an ice cube and to a -40°C

‘Requests for reprints should be addressed to J.-L. Saumet, Laboratoire de Physiologic, Fact&e de Medecine Lyon-Sud, B. P. 12, 69921 Oullins Cedex, France.

NEURONE

;

20-

=

lo-

;:

9.1

O-

: 5

-5

+

*Or 10 0 -5 i 0

20

40 60 60 TIME (sec.) FIG. I. Responses of fiber 9-1 during a series of six stimuli to I5 (a), IO (b), 5 (c). 0 (d). -5 (e) and -5°C (0 again from adapting temperature of 20°C. The afferent spikes were transformed into calibrated impulses of sufficient duration (0.02 set) to allow pen-recording. The frequency of the discharge increased as the intensity of the stimuli increased but it decreased &ghtly for the second stimulation to -.S’-?

NEURONE

5.2

NEURONE

8.1

10

TABLE CHARACTERISTICS

Mechanical threshold (g/mm- ‘)

Fibers

Response ice-cube

I I-I

STUDIED

Response to stimulation at -40°C

to

Conduction velocity (msec -I) 0.8 1.1 3.5 2.6 2.8

YES YES YES YES YES

NO NO YES YES NO

23 23 20 20 30

5-2 8-1 8-2 9-I

1

OF THE UNITS

NEURONE

a 0

5

/,

6

!

-5 5

0

-5

-5

STIMULUS

0 5[

g

3

v)

I \

051 051

NEUW

--

9.1

a

r

I

b

1,

_

L

S[

d

e

-

8” 3

\

10

3--

3 0

”

l

-mm

f -II

li ‘.-

L__1

10

.

\

t

15

t

i

7

5

8.2

0 -5

l

~-1 -5

TEMPERATURE

...‘-._ 1 .__ -5 -5 -5

(“C

-._-_

-5

1

FIG. 2. (A) Threshold course of units 5-2 (O), 8-l (‘.>), 8-2 (A), 9-l (W) during six successive stimuli to 1.5, 10, 5, 0. -5°C and -5°C a second time. (B) Threshold course of unit 11-l during eight successive stimuli to 10°C (once), 0°C (once). and -5°C (six times).

0

1 TIME

2 hn)

i

0 Tk?E

_L__ 2 (mn)

3

FIG. 3A. Averaged post-stimulus frequency histograms during a series of six stimuli to 15°C (a), 10°C (b). 5°C (c), 0°C (d), -5°C (e) and a second time -5°C (f). The bin size was 10 seconds. Adapting temperature before and between the stimulations was 20°C.

SKIN NOCICEPTORS

0

531

AND COLD STIMULATION

1

2

TIME

Imn)

NlnJnoIdE

11.1

3

0

occurred at the adapting temperature. After each stimulation of -5”C, the fibers did not respond to pinching, pin-pricking or radiant heat during a period of 3 to 7 minutes. After this period, nociceptor’s discharge frequencies returned to their initial levels. Discharges of the five units during the cooling stimuli are shown in Fig. 3A for fibers 5-2, 8-I and 9-l and Fig. 3B for fiber 1l-l. Frequency discharge was very small, the m~imum beii 2.2 Hz for unit 1 f-l during -5°C cooling (Fig. 3B). A dynamic discharge occurred only in units 9-l and 1l-l (Fig. 3). Its magnitude was approximately proportional to the intensity of cooling. An increased activity with the repetition of the stimuli was observed in units 5-2, 8-1, 8-2 and 11-l (Fig. 3). 1

2

TIME

(mn)

3

FIG. 3B. Average post-stimulus frequency histograms during eight successive stimuli to 10°C (once) (a), 0°C (once) (b), -5°C (six times) (c, d, e, f, g, h). Adapting temperature before and between the stimulations was 20°C. The bin size was 10 seconds. punctate contact stimulator during 0.4 set was tested and finally, a series of controlled cold stimuli were delivered by a thermode of 6.5 cm2 operating on the Pettier principle 1161 and located so that the nociceptor was as close to the centre of the stimuiator as possible. The skin was adapted for 5 minutes to 20°C (adapting temperature); it was then cooled to 15°C (0.3Y.Xsec), lo”C, 5°C OYJ, -5‘C and a second time to -5”C, (1.3”C/sec); on a single unit the stimulation to -5°C was repeated five times. Each cooling stimulus lasted three minutes. Following each cold stimulus the stimulator was returned to the adapting temperature for 4 minutes. After the thermal stimulation series was completed, the conduction velocity was measured. RESULTS

Twelve receptors in hairy skin have been classified as mechanothermal nociceptors. Their mechanical threshold, measured with the Von Frey hairs, ranged from 20 to 30 g mm-*. Three of them were insensitive to cold. Five others were activated only by a stimulus of -40°C but not by the ice-cube. Four units responded to both stimuli. The cold stimuli were applied to the receptive tields of five nocicep tors. Their general characteristics are shown in Table 1. The complete series of cooling was applied to 4 neurons (5-2, S-1, 8-29-I). The discharge of nociceptor 9-l is shown in Fig. 1. The skin-thermode interface temperature at which the first spike occurred defines the discharge threshold. Since during cooling to IYC, IO??, YC, 0°C -5°C and once again -5”C, the threshold response of fiber 9-l (Fig 1) occurred during the static phase of the stimulus, the observed threshokl is only “apparent” and increased with stimulus temperature, the “actual” threshold was in fact between 20°C and 15°C. The thresholds of fibers 5-2, 8-1, 8-2 and 9-l are shown in Fig. 2 as a function of the stimulus. For fibers 8-l and 8-2 (Fig. 2A) the thresholds were rather similar to those of fiber 9-1. Fibers 5-2 (Fig. 2A) and 1l-1 (Fig. 2B) has “apparent” thresholds proportional to the stimulus temperature for the first, second and third stimuli, since the threshold response occurred during the dynamic phase of the last stimulus; the observed thresholds were above the final temperature. Therefore they represent “actual” thresholds. They increased when stimuli were repeated; for unit 5-2, the threshold increase is so large that a spontaneous activity

DISCUSSION

This preliminary study reports the responses of five cat mechanothermal cutaneous nociceptors to very cold stimuli. These nociceptors had similar responses to cooling. They seem, therefore, to belong to a hom~e~us group. The conduction velocities showed that two units (8-1,5-2) were C fibers and three, units (8-2, 9-1, 11-l) A S fibers. Georgopoulos 110) has also shown that some A 8 fibers, as well as C fibers, responded to thermal and mechanical nociceptive stimuli. Since the classic work of Bessou and Perl [3] the name of polymodal nociceptors is attributed to C nociceptive mechanothermaI~afferents; it seems that this name may be extended to some A 8 nociceptive mechanothermal fibers as well. The mechanical thresholds of these nociceptors, measured by the Von Frey hairs in the hairy skin of the cat, ranged between 20 and 30 g mm-z. The results are close to those of Kumazawa and Per1 1171 who, in a study of 70 nociceptors in the primate hairy skin, found a mechanical threshold ranging between 6 and 26 g mm-*. However, the mean threshold computed from 34 nociceptive fibers in the monkey glabrous skin [ 191 and from 87 rnec~o~e~~ units in the glabrous and hairy skin of the monkey [20] was equal to 60 g rnrnw2 and46 g rnrnez respectively. Those results allow us to speculate that mechanical thresholds ate smaller in hairy skin than in glabrous one and probably lower in cat than in monkey. Our study shows that some mechanothermal nociceptors are activated by very cold stimuli. This result confirms previous observations [19,21] which showed that mechanothermal’ nociceptors may be activated by cooling due to the evaporation of ether [21] or by contact with an ice-cube 119,201.. Our study shows that the response thresholds of these fibers to cooling in unsensitized fibers increases with the decrement of the stimulus temperature, since the fmt action potential was noted during the steady phase of the temperature change. The latency of the response was more than twenty seconds. This may be expIained by a deep position of nociceptors in the skin or by their relatively low sensitivity to cold. Repetition of very cold stimulations decreases the discharge threshold of these nociceptors. This_;.phenomenon is known to occur also in mechanothermal nociceptors excited by noxious heat 9.91 and chemical stimuli fill. It may explain the hy~~sthesia observed in humans when noxious stimuli are repeated. In Georgopoulos’s [ 12,133 and LaMotte’s [20] studies as in ours the discharge intensity increased in proportion to the stimulus intensity. We have observed that immediately after the application for 3 minutes of the -5°C stimulus, the mechanothermal nociceptors were completely inhibited for 3 to 7 minutes. This inhibition was probably due to an ‘inac-

tivation of the neuron metabolism by cold. The phenomenon may explain why cold is used as a local anaesthetic in human therapeutics. This may also explain why cutaneous freezing may occur in humans without any prior indication of pain. However, this interpretation may not be the only one since other cutaneous receptors respond to very cold temperatures. In a recent paper [lo], 47 cold fibers have been studied in cats’ noses. Among these, 2 fibers showed an increasing frequency when the cutaneous temperature fell from I S’C to P5”C. Moreover, LaMotte and Thalhammer 1201 have observed in monkey that the total number of impulses of 7 high threshold cold receptors increased monotonically with decreasing stimulus temperature. The responses of the fibers studied by Georgopoulos [12.13] increased with the stimulus intensity as a power function. Therefore, mechanothermal

nociceptors and cold receptors could signal the noxwu\ne\~ of cold pain. However. in humans. it has been shown that punctate burning, punctate freezing and needle pricking arc felt as a pricking sensation 171 whilst the cold nature of the stimulus may be detected only when the stimulator ha\ an areas of 3 mm 121. Since some mechanothermal nociceptors are polymodal, in the sense that they respond to all these noxious stimuli, they probably have an important function in the coding of the perception threshold of cold pain. The cold quality of the stimulus would be encoded by high threshold cold receptors as suggested by LaMotte and Thalhammet [20], but equally by “common-type” cold receptors which are certainly activated by the diffusion of cold in the tissue [ 5.71 surrounding the stimulating device and are all the more numerous as the stimulator aiLc is greater

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