Do Fish have Nociceptors? Evidence for the Evolution of a Vertebrate Sensory System

Abstract

Nociception is the detection of a noxious tissue-damaging stimulus and is sometimes accompanied by a reflex response such as withdrawal. Pain perception, as distinct from nociception, has been demonstrated in birds and mammals but has not been systematically studied in lower vertebrates. We assessed whether a fish possessed cutaneous nociceptors capable of detecting noxious stimuli and whether its behaviour was sufficiently adversely affected by the administration of a noxious stimulus. Electrophysiological recordings from trigeminal nerves identified polymodal nociceptors on the head of the trout with physiological properties similar to those described in higher vertebrates. These receptors responded to mechanical pressure, temperatures in the noxious range (more than 40 degrees C) and 1% acetic acid, a noxious substance. In higher vertebrates nociceptive nerves are either A-delta or C fibres with C fibres being the predominating fibre type. However, in the rainbow trout A-delta fibres were most common, and this offers insights into the evolution of nociceptive systems. Administration of noxious substances to the lips of the trout affected both the physiology and the behaviour of the animal and resulted in a significant increase in opercular beat rate and the time taken to resume feeding, as well as anomalous behaviours. This study provides significant evidence of nociception in teleost fishes and furthermore demonstrates that behaviour and physiology are affected over a prolonged period of time, suggesting discomfort.

Full text

1
2
3
4
FirstCite
e-publishing
5
12345678910111213141523333435
Received 5 December 2002
Accepted 15 January 2003
36
Published online
4048
495051
52
Do fishes쐌쐌9쐌쐌 have nociceptors: evidence for the
53 evolution of a vertebrate sensory system
54 Lynne U. Sneddon
1
†, Victoria A. Braithwaite
2
and Michael J. Gentle
55 쐌쐌1쐌쐌
1
56
1
Roslin Institute, Welfare Biology, Roslin, Midlothian EH25 9PS, UK
57
2
Division of Biological Sciences, Ashworth Laboratories, University of Edinburgh EH9 3JT, UK
58
Nociception is the detection of a noxious, tissue damaging stimulus and is sometimes accompanied by a
59
reflex response such as withdrawal. Pain perception, as distinct from nociception, has been demonstrated
60
in birds and mammals but has not been systematically studied in lower vertebrates. We assessed whether
61
a fish possessed cutaneous nociceptors capable of detecting noxious stimuli and if its behaviour was suf-
62
ficiently adversely affected by administration of a noxious stimulus. Electrophysiological recordings from
63
trigeminal nerves identified polymodal nociceptors on the head of the trout with physiological properties
64
similar to those described in higher vertebrates. These receptors responded to mechanical pressure, tem-
65
peratures in the noxious range (more than 40 °C) and 1% acetic acid, a noxious substance. In higher
66
vertebrates nociceptive nerves are either A-delta or C fibres with C fibres being the predominating fibre
67
type. However, in the rainbow trout A-delta fibres were most common, and this offers insights into the
68
evolution of nociceptive systems. Administration of noxious substances to the lips of the trout affected
69
both physiology and behaviour of the animal and resulted in a significant increase in opercular beat rate
70
and the time taken to resume feeding, as well as anomalous behaviours. This study provides significant
71
evidence of nociception in teleost fishes and furthermore demonstrates that behaviour and physiology are
72
affected over a prolonged period of time suggesting discomfort.
73
Keywords: nociception; pain; rainbow trout; trigeminal
74
75
76
1. INTRODUCTION
77
Nociception, the detection of tissue damaging stimuli, is
78
evident in a number of different phyla including birds and
79
mammals (Walters 1996), but studies on lower vertebrates
80
have suggested a lack of nociceptors and pain perception
81
(e.g. Atlantic stingray (Dasyatis sabina), Coggeshall et al.
82
(1977쐌쐌2쐌쐌) and Leonard (1985); or long-tailed stingray
83
(Himantura fai ), Snow et al. (1993)). From the perspec-
84
tive of the evolution of sensory function in vertebrates, the
85
study of sensory systems in lower vertebrates is of great
86
interest. Olfactory, gustatory and chemosensory systems
87
have been well described in fishes (Belousova et al. 1983;
88
Kotrschal 2000), but relatively little attention has been
89
paid to nociception. The trigeminal nerve, the fifth cranial
90
nerve, innervates the majority of sensory information from
91
the head of vertebrates and as such conveys somatosensory
92
information from potentially damaging stimuli to the
93
brain. A study on the most primitive living vertebrate, the
94
lamprey (Petromyzon marinus), suggested that there were
95
trigeminal receptors that responded to burning of the skin
96
(Matthews & Wickelgren 1978). The physiological
97
responses of these receptors, however, were not well
98
characterized and the responses recorded may have been
99
a result of damage to the receptor field rather than the
100
preferential sensitivity to a noxious temperature per se.
101
Furthermore, the lamprey lacks myelination, and its clos-
102
est evolutionary group, the elasmobranchs, are deficient
1
30
† Present address: University of Liverpool, School of Biological Sciences,
31
BioScience Building, Liverpool L69 3BX, UK (lsneddon@liverpool.ac.
32
uk).
1
2
Proc. R. Soc. Lond. B 02PB1068.1  2003 The Royal Society
3
DOI 10.1098/rspb.2001.2349
6
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
103
in unmyelinated fibres and no nociceptors have been
104
identified (Leonard 1985; Snow et al. 1993). A recent
105
study on the rainbow trout (Oncorhynchus mykiss) demon-
106
strated that, although most primary afferent somatosen-
107
sory fibres were A-delta fibres, unmyelinated C fibres were
108
present in the trigeminal nerve (Sneddon 2002). Free
109
nerve endings of A-delta and C fibres act as nociceptors in
110
higher vertebrates and have been well characterized (Lynn
111
1994) and thus there is the potential for these neurons to
112
act as nociceptors in the rainbow trout.
113
A number of different classes of nociceptors have been
114
described in mammals but they are commonly slowly
115
adapting mechanoreceptors that preferentially respond to
116
noxious heat (greater than 40 °C) and are termed mechan-
117
othermal nociceptors (Lynn 1994). If these nociceptors
118
also respond to noxious chemicals such as bee venom,
119
acid, bradykinins, acetyl choline, then they are classified
120
as polymodal nociceptors (Lynn 1994). Using electrophy-
121
siological techniques, nociceptors have been identified in
122
amphibia (Spray 1976쐌쐌3쐌쐌), birds (Gentle 1992, 1997;
123
Gentle & Tilston 2000), mammals (Yeomans & Proudfit
124
1996) including primates (Kenshalo et al. 1989) and
125
humans (Torebjo
¨
rk & Hallin 1974; Hallin et al. 1981).
126
Therefore, if we can demonstrate that the rainbow trout
127
possesses the neural apparatus to detect noxious stimuli,
128
then this confirms that the trout is capable of nociception,
129
the simple detection and reflex response to a noxious
130
stimulus (Kavaliers 1988; Bateson 1991쐌쐌4쐌쐌). To sug-
131
gest pain perception, it must be shown that any behav-
132
ioural or physiological responses are not merely reflexive.
133
Pain in humans has been defined as an ‘unpleasant sen-
134
sory and emotional experience associated with actual or

123
1
02PB1068.2 L. U. Sneddon and others Nociception in fishes
2
3
135
potential tissue damage’ (IASP 1979쐌쐌5쐌쐌). It is imposs-
136
ible to truly know if an animal has an emotion because we
137
cannot measure emotion directly. Therefore, emotion
138
does not feature in the definition of pain in animals
139
(Zimmerman 1986; Bateson 1991쐌쐌4쐌쐌). What an ani-
140
mal ‘feels’ is possibly nothing like the experience of
141
humans with a more complex brain structure, however,
142
the animal’s experience may be unpleasant or cause suffer-
143
ing and their discomfort is no less important in terms of
144
biology or ethics. To examine possible pain perception in
145
an animal, indirect measurements of behavioural and
146
physiological responses to a potentially painful event are
147
made and then we decide upon the evidence collected
148
from the data as is routinely done in welfare studies
149
(Bateson 1991쐌쐌4쐌쐌; Broom 1991; Gentle 1992; Gonyou
150
1994; Bradshaw & Bateson 2000; Mason et al. 2001;
151
Roughan & Flecknell 2001; Molony et al. 2002). If a
152
noxious event has sufficiently adverse effects on behav-
153
iour and physiology in an animal and this experience is
154
painful in humans, then it is likely to be painful in the
155
animal.
156
To demonstrate that an animal is capable of pain per-
157
ception, it must first perceive the adverse sensory stimulus
158
and then react both physiologically (e.g. inflammation,
159
cardiovascular changes) and behaviourally (move away
160
from stimulus). However, to show that this is not simply
161
a nociceptive reflex, it is necessary to show that the animal
162
learns that the stimulus is associated with an unpleasant
163
experience and avoids it. Certainly it has been demon-
164
strated that fishes can learn to avoid an adverse stimulus
165
such as electric shock (Ehrensing et al. 1982) and hooking
166
during angling (Beukema 1970a,b). Additionally, suffer-
167
ing or discomfort is implicated if the animal’s behaviour
168
is adversely affected (Zimmerman 1986). These criteria
169
have been demonstrated in mammals (Roughan & Fleck-
170
nell 2001), birds (Gentle 1992) and amphibians (Stevens
171
1992) but not in teleost fishes.
172
The purpose of the present study was to determine if
173
nociceptors were present in the trigeminal system on the
174
head of the trout and to determine the physiological and
175
behavioural consequences of prolonged noxious stimu-
176
lation. Recordings were made from the trigeminal nerve
177
to identify if nociceptors were present on the face and
178
head of the trout. Behavioural and physiological responses
179
of the fish to administration of acutely algogenic sub-
180
stances to the lips of the trout were assessed to examine
181
if there was the potential for pain perception in this spec-
182
ies. The criteria that must be met for animal pain are first,
183
the demonstration of the sensory capability of detecting
184
potentially painful stimuli, and second, the performance
185
of adverse behavioural responses to a potentially painful
186
event that are not simple reflexes.
187
2. METHODS
188
(a) Electrophysiological recordings from the
189
trigeminal ganglion
190 Rainbow trout (750 ± 100 g, n = 10) were supplied by a com-
191 mercial fish supplier. The fishes were maintained as described
192 in a previous study (Sneddon 2002). Trout were caught indi-
193 vidually by netting and initially anaesthetized by immersion in
194 MS 222 (50 mg l
⫺1
) to facilitate weighing and intraperitoneal
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
195
injection of Saffan (0.3 ml 100 g
⫺1
; Schering-Plough Animal
196Health, Welwyn Garden City, UK). Once deep anaesthesia was
197achieved, the fish was placed into a stainless steel cradle
198cushioned with wet paper towels and held in position with
199Velcro straps. The fishes had reached surgical, deep plane anaes-
200thesia and were not conscious and had to be ventilated by flush-
201ing fresh water over the gills by means of a tube held in place
202by a specially constructed mouth piece. Skin and bone were
203removed above the brain and then the olfactory and optic lobes
204and cerebellum were removed via a suction tube connected to
205a vacuum pump. This procedure is known as decerebration and
206renders the animal insentient because it is only left with a brain
207stem. To prevent muscular twitching, Pavulon, a neuromuscular
208blocker (pancurorium bromide 2 mg ml
⫺1
), was injected intra-
209muscularly (0.08 ml 100 g
⫺1
fish weight). Bone was removed to
210expose the trigeminal ganglion and the ganglion was desheathed
211and covered in paraffin to prevent moisture loss. Glass insulated
212tungsten microelectrodes (tip diameter 10 µm) were used to rec-
213ord from afferent cell bodies. The extracellular action potentials
214were amplified using a NL100 head stage connected to a NL104
215preamplifier (Neurolog System, Digitimer Ltd, UK). The signal
216was displayed on a storage oscilloscope (5113, Tektronix INC)
217and stored on a PC using a Micro 1401 interface and Spike 2
218software (CED, UK).
219Neural activity was recorded from single cells in the trigeminal
220ganglion following the application of stimuli to the head of the
221fish. A glass mechanical probe (0.1 mm diameter) was lightly
222applied to the facial skin in order to locate a receptor field. Once
223located, the mechanical threshold of the receptor was determ-
224ined by applying von Frey hairs (0.1–15.0 g at 0.1 g intervals)
225to the receptor field. The diameter of the receptive field was
226measured to 0.1 mm using Vernier calipers. The receptor was
227then tested for thermal and chemical sensitivity. A thermal
228stimulator was placed 1 mm above the area of the receptor field
229so that it did not burn the skin and the stimulator raised the
230temperature to 58 °C. Thermal sensitivity was determined by
231heating the skin at a rate of 1 °Cs
⫺1
up to 58 °C using a prefo-
232cused quartz glass light bulb with built in reflector (A1231,
23312 V, 100 W Wotan) orientated vertical to the skin. If the recep-
234tor responded to the increase in temperature, the threshold was
235determined and the response had to be repeatable. Temperature
236was measured using a type K thermocouple placed in the centre
237of the bulb focus and was controlled by a feedback circuit. The
238skin temperature was held at 58 °C for 10 s after which it rapidly
239returned to normal. The temperature increase of 1 °Cs
⫺1
240allowed the threshold to be determined. To ascertain chemosen-
241sitivity, a drop of 1% acetic acid was placed onto the receptor
242field. The first 5 ms after the addition of the drop was disre-
243garded as this could be a response to the touch of the drop; a
244response to this noxious chemical stimulation was confirmed if
245the action potentials measured from mechanical and/or thermal
246stimulation of that receptor fired after this period. Again this
247response was repeatable. A drop of water was also placed onto
248the receptive field to act as a control stimulus. None of the
249receptors responded to this. Conduction velocities were
250obtained by placing silver wire stimulation electrodes onto the
251receptor field, and stimulating the receptor directly by an electri-
252cal pulse. This stimulated the fibre to produce an action poten-
253tial and the conduction velocity was determined using the time
254that the action potential was recorded after the stimulus and
255the estimated distance travelled from the receptive field to the
256recording electrode in the trigeminal ganglion.

123
1
Nociception in fishes L. U. Sneddon and others 02PB1068.3
2
3
257
(b) Behavioural responses to administration of
258
algogenic substances
259 Twenty rainbow trout (30–100 g) were obtained from a com-
260 mercial fish supplier, individually housed in rectangular tanks
261 (45 cm × 25 cm × 35 cm) with a constant flow of water at
262 11 ± 1 °C and a feeding ring (10 cm diameter) secured on the
263 water surface at the same location in each tank. One half of the
264 tank was covered by an opaque lid (22.5 cm × 25 cm) to provide
265 an area of shelter, whereas the other half had a transparent lid
266 and this was where the feeding ring was located. Each tank had
267 a gravel substrate and was continuously aerated via an airstone
268 and tubing connected to an air pump. Each fish was trained
269 twice daily, am and pm, to come to the ring to receive food
270 pellets (TROUW Aquaculture, UK) in response to a light cue
271 above the tank (one test equals one trial; mean number of trials
272 to learn, 10 ± 4). Once the fishes had learned to feed at the ring
273 by successfully performing six consecutive trials they received
274 two weeks’ further training to ensure that they were truly con-
275 ditioned to the light stimulus (i.e. responded to light only before
276 food presentation and they had to perform another 14 trials suc-
277 cessfully to be included in the experiment). Fishes were then
278 assigned to four treatment groups: (i) saline—0.1 ml sterile
279 saline injected (25 g needle and 1 ml syringe) into frontal lips;
280 (ii) venom—0.1 ml bee venom (1 mg ml
⫺1
sterile saline) injected
281 into frontal lips; (iii) acid—0.1 ml acetic acid (0.1% in sterile
282 saline) injected into frontal lips; and (iv) control—fish handled
283 but received no injection.
284 Acetic acid and bee venom were chosen because the protons
285 of the acid stimulate nociceptive nerves in mammals (Martinez
286 et al. 1999) and frogs (Hamamoto et al. 2000), and the venom
287 has an inflammatory effect in mammals (Lariviere & Melzack
288 1996) and both are known to be painful in humans. Before treat-
289 ment the behaviour and opercular (gill) beat rate were measured
290 continuously for 15 min. Behaviours recorded were their pos-
291 ition in tank (under covered or exposed area) and swimming
292 activity (direct movement of fishes more than one body length).
293 Fishes were then individually anaesthetized using benzocaine
294 (1.5 ml (50 mg l
⫺1
ethanol) l
⫺1
) and were carefully injected with
295 the appropriate substance into the upper and lower frontal lip
296 or handled but not injected. The fishes were in medium to deep
297 plane anaesthesia during this procedure and had lost all reflex
298 activity and muscular control. Trout were placed back into their
299 original tank and allowed 30 min to recover from the anaes-
300 thesia. Behaviour and opercular beat rate were recorded for
301 15 min and then the light was switched on and food sub-
302 sequently introduced to the tank. If the fishes did not respond
303 by swimming to the feeding ring to feed they were left for a
304 further 30 min, then a further 15 min of observations were
305 recorded and light cue and food given. This regime continued
306 until the fishes resumed feeding. All fishes ingested food within
307 ca. 4 h. The time to perform the feeding ring task and resume
308 feeding for the four groups was compared using one-way
309 ANOVA. The percentage of time spent in the covered area for
310 each fish in all four groups was determined before and after the
311 treatment and compared using Mann–Whitney U-tests. Fre-
312 quency of swimming activity was calculated for each fish in the
313 experimental groups and before and after the treatment, and also
314 compared using Mann–Whitney U-tests.
315 In a second experiment, six rainbow trout were trained as
316 described above, however, half of these were fed live red mos-
317 quito larvae instead of pellets to provide a softer foodstuff. All
318 fishes were injected with bee venom and assessed for behaviour
319 and opercular beat rate as already described. The time to resume
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
728
729
730
731
732
Figure 1. Position of polymodal mechanoreceptors or
733
nociceptors, mechanothermal receptors and
734
mechanochemical receptors on the head and face of the
735
rainbow trout, Oncoryhnchus mykiss (triangles, polymodal
736
nociceptor; diamonds, mechanothermal nociceptor;
737
hexagons, mechanochemical receptor).
320feeding on the two different diets was compared using a Krus-
321kal–Wallis test due to the low sample size, which was chosen for
322ethical reasons.
323All the fishes used in both experiments were held for a further
3243 days and trained in the conditioning task twice a day. All fishes
325continued to successfully perform the task and ingest food,
326therefore, there appeared to be no chronic effects on associative
327learning and appetite. At the end of the 3 days, the trout were
328individually killed by overdose in anaesthetic.
329
3. RESULTS
330
(a) Characterization of nociceptors
331
Fifty-eight receptors were located on the face and head
332
of the rainbow trout. Twenty-two of these receptors could
333
be classified as nociceptors (figure 1) as they responded
334
to mechanical pressure by a slowly adapting firing pattern
335
and were also stimulated by noxious heat stimulation
336
(more than 40 °C) and of these, 18 also responded to
337
algogenic chemical stimulation (1% acetic acid; figure 2a–
338
c). The response of the receptors to mechanical, noxious
339
thermal and chemical stimulation clearly characterizes
340
them as polymodal nociceptors (table 1). There were four
341
receptors that did not respond to chemical stimulation and
342
are classified as mechanothermal nociceptors. A third
343
group of receptors (n = 6) responded to only mechanical
344
and chemical stimulation, but without a detailed investi-
345
gation of their physiological characteristics they cannot be
346
classified as nociceptors at present and are referred to as
347
mechanochemical receptors. A further 16 receptors gave
348
a slowly adapting response to mechanical stimulation and
349
another 14 receptors gave a rapidly adapting response, but
350
none of these responded to thermal or chemical stimu-
351
lation and are possibly pressure and touch receptors,
352
respectively (Sneddon 2003). The characteristics of the
353
polymodal and mechanothermal nociceptors and the
354
mechanochemical receptors are shown in table 1. Mech-
355
anical thresholds of the three types ranged between 0.1
356
and 7.1 g and conduction velocities were recorded

123
1
02PB1068.4 L. U. Sneddon and others Nociception in fishes
2
3766
767
Table 1. Characteristics of the three types of receptor found on the head of the rainbow trout. Values shown are means ± s.e.
768
773
778
783
polymodal nociceptors mechanothermal nociceptors
(n = 18) (n = 4) mechanochemical receptors (n = 6)
790
795
800
805
diameter of receptor 2.52 ± 0.4
(mm) 3.20 ± 0.4 2.83 ± 1.0
811
mechanical threshold (g) 0.83 ± 0.4 0.1 ± 0.0 0.78 ± 0.53
816
thermal threshold (°C) 49.3 ± 1.4 46.2 ± 2.4 none
821
acid response yes none yes
826
conduction velocity
(m s
⫺1
) 3.96 ± 0.4 3.71 ± 0.5 4.28 ± 0.1
832
837
739
740
741
on
1 s
60
15
˚C
2 s
acid
1 s
60
15
5 s
(a)
(b)
(c)
(d )
˚C
742
743
Figure 2. A polymodal nociceptor responding to (a)
744
mechanical, (b) thermal and (c) chemical stimulation (1%
745
acetic acid). The receptor is slowly adapting to mechanical
746
stimulation (a)(‘on’ indicates application of stimulus), has a
747
thermal threshold of 58 °C(b), and responds to application
748
of a drop of acetic acid onto the receptive field (c). (d )A
749
polymodal nociceptor with a thermal threshold of 42.3 °C.
357
between 0.97 and 8.5 m s
⫺1
. Out of all the polymodal
358
nociceptors that were recorded from, only one was a
359
unmyelinated C fibre and the rest were A-delta. Thermal
360
responses were only seen above 40 °C and thresholds
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
361
ranged from 40 °Cto58°C (figure 2b,d). The diameter
362
of the receptor field ranged from 1.6 to 9 × 1 mm. Inter-
363
estingly, we found no thermal receptors that responded to
364
temperature in the range of 20 °Cto40°C.
365
(b) Behavioural and physiological responses to
366
acute noxious stimulation
367
Significant increases in opercular beat rate were found
368
in all four groups after the treatment (control and saline:
369
ca. 52 beats min
⫺1
to 70 beats min
⫺1
) although the venom
370
and acid groups had greatly elevated rates after the treat-
371
ment (ca. 52 beats min
⫺1
before to 93 beats min
⫺1
after
372
treatment; figure 3a; F
3,16
= 27.52, p ⬍ 0.001). This
373
physiological effect was also coupled with profound effects
374
on the fishes’ behaviour. It took control and saline fishes
375
ca. 80 min to begin ingesting food again whereas venom
376
and acid fishes took ca. 170 min (figure 3b; F
3,16
= 7.29,
377
p = 0.003). In addition to this, we performed the second
378
experiment that tested whether the fishes would resume
379
feeding more quickly if fed on a softer foodstuff but there
380
was no significant difference in the time to resume feeding
381
(H = 0.05, p = 0.827, d.f. = 1).
382
Activity levels were not affected by the treatment
383
whether it was potentially painful (W = 130.5, p = 0.057)
384
or not (W = 107.0, p = 0.908; median frequency before
385
= 0.356 min
⫺1
; after = 0.326 min
⫺1
) although there was a
386
trend for the venom and acid injected fishes to reduce the
387
amount of swimming activity (median frequency before
388
= 0.935 min
⫺1
; median frequency after = 0.265 min
⫺1
).
389
Position in tank or use of the sheltered area was also not
390
affected by the noxious injections (W = 103, p = 0.910;
391
median percentage time spent under cover before
392
= 53.3%; after = 55.8%) or the controls treatments
393
(W = 106; p = 0.970; before = 53.9%; after = 63.0%).
394
Observations following acid and venom injection found
395
that the fishes performed anomalous behaviours after the
396
treatment that were not seen in the control or saline
397
groups; acid and venom fishes performed ‘rocking’ where
398
the fishes moved from side to side balancing on either pec-
399
toral fin while resting on the gravel (mean frequency
400
0.37 min
⫺1
for venom group and 0.45 min
⫺1
for acid
401
group). The acid group was also observed to rub their lips
402
into the gravel and against the tank walls but the venom
403
group did not perform this behaviour.
404
4. DISCUSSION
405
The polymodal nociceptors found here in the trout have
406
similar properties to those found in amphibians (Stevens
407
1992), birds (Gentle 1992, 1997) and mammals

123
1
Nociception in fishes L. U. Sneddon and others 02PB1068.5
2
3751
752
753
100
75
50
250
200
150
100
50
0
1
2
3
4
5
6
control
saline
venom
acid
mean time to resume feeding (min)
opercular beat rate
time
(a)
(b)
754
755
Figure 3. (a) Mean (± s.e.m.) opercular beat rate of each
756
treatment group 20 min before treatment and at each
757
observation afterwards (time 1 is 20 min before treatment;
758
time 2 is 30 min after treatment and each time point after
759
this is ca. 30 min apart). (Grey dashed line, control; black
760
solid line, saline; grey solid line, venom; black dashed line,
761
acid.) (b) The mean (± s.e.m.) time taken for each fish in
762
each treatment group to resume ingesting food after the
763
treatment.
408
(Handwerker et al. 1987) including humans (Lynn 1994).
409
Nociceptors, by definition, preferentially respond to noxi-
410
ous, injurious stimuli and this demonstrates that the rain-
411
bow trout is capable of nociception (Kavaliers 1988;
412
Bateson 1991쐌쐌4쐌쐌). Receptor diameter, thermal thresh-
413
olds and mechanical responses are similar to those meas-
414
ured in higher vertebrate groups (Torebjo
¨
rk & Hallin
415
1974; Spray 1976쐌쐌3쐌쐌; Hallin et al. 1981; Kenshalo et al.
416
1989; Yeomans & Proudfit 1996; Gentle & Tilston 2000).
417
Mechanical thresholds were lower than those found in
418
humans; at least 0.6 g is required for noxious stimulation
419
in human skin (Lynn 1994) and many of the nociceptors
420
on the fish skin were stimulated by 0.1 g. This may be due
421
to the more easily damaged nature of the fish skin and as
422
such the nociceptors have lower thresholds. Similar thre-
423
sholds were found in mammalian eye nociceptors
424
(Belmonte & Gallar 1996) and so the fish nociceptors
425
have mechanical thresholds comparable with those in the
426
cornea of the eye.
427
None of the trigeminal receptors in this study was
428
stimulated by temperatures in the range of 30 °Cto40°C.
429
A number of studies have demonstrated a lack of thermal
430
receptors in invertebrates and other lower vertebrates
431
(Matthews & Wickelgren 1978; Leonard 1985; Walters
432
1996). This suggests that thermal receptors in the non-
433
noxious range potentially evolved in vertebrate groups that
434
lead a more terrestrial existence. These thermal receptors
435
may have evolved in response to temperature fluctuations
436
in the terrestrial environment. It is unlikely that the rain-
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
437
bow trout would come into contact with such high noxi-
438
ous heat as used in this study and this species inhabits
439
waters below 25 °C. The nociceptors of this fish respond
440
only above 40 °C and this is typical of nociceptors in
441
higher vertebrates. This would suggest that either in the
442
distant evolutionary past the animals encountered tem-
443
peratures above 40 °C, or the response to such high tem-
444
peratures may be a fundamental physiological mechanism
445
or property of nociceptive nerve endings, as has been dem-
446
onstrated in rat cultured dorsal root ganglion neurons
447
(Lyfenko et al. 2002). These dorsal root neurons would
448
also not come into direct contact with noxious tempera-
449
tures, but they are responsive only to temperatures in the
450
noxious range. It would be interesting from a comparative
451
point of view to assess nociceptive responses in a tropical
452
fish species because they would encounter higher tempera-
453
tures. The mechanochemical receptors did not respond to
454
thermal stimulation and cannot be classified as nocicep-
455
tors. Further work is required to test these receptors with
456
a variety of chemicals to ascertain if these are simply
457
chemoreceptors, or if they are nociceptive, they only
458
respond to noxious chemicals.
459
Assessing the subjective experiences of animals plays an
460
increasingly large role in animal welfare (Broom 1991;
461
Gentle 1992; Dawkins 1998; Bradshaw & Bateson 2000;
462
Mason et al. 2001). To date, little attention has been paid
463
to potential pain perception in fishes. In our behavioural
464
experiments, we trained fishes to come to a feeding ring
465
in response to a light cue and then assigned them to four
466
treatment groups; three of these groups had either bee
467
venom, acetic acid or saline injected into the lips and a
468
fourth group was simply a handled control. After injection
469
of algogenic substances, the resulting increase in opercular
470
rate is similar to that recorded when trout are swimming
471
at maximum speed (Altimiras & Larsen 2000) and much
472
greater than the rate recorded after handling stress
473
(increase to a maximum of 69 beats min
⫺1
(Laitinen &
474
Valtonen 1994)). The control and saline groups showed
475
similar increases in opercular beat rate to stressed fishes
476
(Laitinen & Valtonen 1994) and this is probably due to
477
the handling and anaesthetic procedure. Respiratory
478
changes have been demonstrated in mammals and
479
humans enduring a nociceptive event (Kato et al. 2001)
480
and so this dramatic rise in ventilation rate may be a
481
physiological response to noxious stimulation in the rain-
482
bow trout.
483
The rainbow trout injected with acetic acid and bee
484
venom performed anomalous behaviours that were not
485
performed by the saline or control groups. Rocking behav-
486
iour was seen in both venom and acid treatment groups
487
and this behaviour was performed only in the 1.5 h after
488
injection. This is reminiscent of the stereotypical rocking
489
behaviour of primates that is believed to be an indicator
490
of poor welfare and thought to be performed as a comfort
491
behaviour (Gonyou 1994). The performance of anomal-
492
ous behaviours usually occurs within a short time period
493
after the occurrence of a painful event when the pain is
494
most intense (Molony et al. 2002). Only the acid group
495
performed rubbing of the lips against the gravel and the
496
sides of the tank. The act of rubbing an injured area to
497
ameliorate the intensity of pain has been demonstrated in
498
humans and in mammals (Roveroni et al. 2001). Overall,
499
the administration of noxious substances had a negative

123
1
02PB1068.6 L. U. Sneddon and others Nociception in fishes
2
3
500
affect on the fishes’ behaviour. To our knowledge, the per-
501
formance of these behaviours has not been observed in
502
fishes before. These behaviours may be indicative of dis-
503
comfort and may have a potential use as indicators of pain
504
or the occurrence of a noxious event in fishes. However,
505
in humans and other animals pain is a specific experience
506
and each different type of pain may have different behav-
507
ioural responses and may also be species specific
508
(Kavaliers 1988). Therefore, further studies should target
509
noxious stimulation of other areas of the fish body to
510
assess whether the behaviours seen in this study are uni-
511
versal.
512
The venom and acid injected fishes took ca. 3 h to begin
513
ingesting food, whereas the saline and control groups took
514
ca. 1 h. The venom and acid groups may be experiencing
515
discomfort and so take longer to perform the task and
516
resume feeding. This may be similar to guarding behav-
517
iour where an animal does not use a painful limb to pre-
518
vent more pain and damage being caused to the affected
519
area (Gentle 1992). Handling and anaesthesia are known
520
to be stressful, causing an elevation in respiration rate
521
(Laitinen & Valtonen 1994) and would account for the
522
delay in the saline and control groups to perform the con-
523
ditioning task. Giving the noxiously stimulated trout softer
524
foodstuff did not affect the time to begin feeding again.
525
Therefore, it appears as if the rainbow trout does not feed
526
when affected by the administration of a noxious agent to
527
the lips and only resumes feeding when the behavioural
528
and physiological effects subside.
529
Our results demonstrate that the rainbow trout pos-
530
sesses nociceptors that detect noxious stimuli and that
531
both the behaviour and physiology of the rainbow trout
532
are adversely affected by stimuli known to be painful to
533
humans. The behaviours shown by the trout after injection
534
of a noxious stimulus are complex in nature and as such
535
may not be simple reflexes. The performance of rocking
536
behaviour and rubbing of the affected area, possible indi-
537
cators of discomfort, suggests that higher processing is
538
involved in the behavioural output and this is similar to
539
some of the responses of higher vertebrates (Gonyou
540
1994; Roughan & Flecknell 2001) and man (Kato et al.
541
2001) to noxious stimuli. Other behavioural studies have
542
shown that fishes learn to avoid aversive, noxious events
543
such as electric shock but fishes that had morphine, an
544
analgesic, administered failed to learn to avoid the electric
545
shock (Ehrensing et al. 1982). Together, these electrophy-
546
siological and behavioural results show that the rainbow
547
trout has a well developed nociceptive system. Previous
548
anatomical studies have suggested marine elasmobranches
549
do not have nociceptors (Leonard 1985; Snow et al.
550
1993). This may represent an evolutionary divergence
551
between the teleost and elasmobranch lineages.
552
Interestingly, there is a higher percentage of A-delta
553
fibres (25%) in the trigeminal nerve compared with C
554
fibres (4%; Sneddon 2002) and the majority of nocicep-
555
tors were recorded from A-delta fibres. Only one of the 18
556
nociceptors we recorded from had a conduction velocity in
557
the range of C fibre velocity (0.97 m s
⫺1
) and the rest were
558
A-delta fibres. Studies in mammals have stressed the
559
importance of C fibres in prolonged nociceptive stimu-
560
lation because they act as polymodal nociceptors with A-
561
delta fibres, being mechanothermal nociceptors, partici-
562
pating only in acute short-term responses usually to alert
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
563
the nervous system to immediate injury (Matzner & Devor
564
1987; Lynn 1994; Gentle 1997). However, A-delta fibres
565
predominate in the rainbow trout and the behavioural
566
effects of a noxious stimulus, such as bee venom, were
567
prolonged over ca. 3 h. Therefore, in teleosts, A-delta
568
fibres potentially have a dual role in mediating reflex
569
escape behaviour as well as prolonged noxious stimu-
570
lation, whereas in higher vertebrates, C fibres may have
571
evolved to become more numerous and have a more
572
prominent function in prolonged noxious stimulation and
573
inflammatory pain. More detailed electrophysiological
574
recordings on A-delta fibres in the trout are necessary to
575
confirm this hypothesis. Sneddon (2002) suggested that
576
the higher proportion of C fibres in the higher vertebrates
577
compared with the teleost was due to the advance onto
578
land in evolution and the increased chance of injury due to
579
gravity, extremes of temperature and noxious gases. The
580
aquatic environment provides buoyancy, dilution of
581
chemicals and a relatively stable thermal environment and
582
so perhaps teleosts have not dedicated such a great
583
amount of neural wiring to nociception as terrestrial ver-
584
tebrates have.
585
The results of the present study demonstrate nocicep-
586
tion and suggest that noxious stimulation in the rainbow
587
trout has adverse behavioural and physiological effects.
588
This fulfils the criteria for animal pain as stated in § 1.
589
Future work should examine the cognitive aspects of noxi-
590
ous stimulation to assess how important enduring a noxi-
591
ous, potentially painful event is to the mental well-being
592
of this species.
593
We are grateful to BBSRC for funding (grant no. 215/S11042).
594
L.U.S. thanks Jon Banks, University of Manchester, for his
595
advice on the electrophysiology.
596
REFERENCES
597
Altimiras, J. & Larsen, E. J. 2000 Non-invasive recording of
598
heart rate and ventilation rate in rainbow trout during rest
599
and swimming: fish go wireless! J. Fish Biol. 57, 197–209.
600
Bateson, P. 1992 Assessment of pain in animals. Anim. Behav
601
42, 827–839. 쐌쐌4쐌쐌.
602
Belmonte, C. & Gallar, J. 1996 Corneal nociceptors. In Neuro-
603
biology of nociceptors (ed. C. Belmonte & F. Cervero), pp.
604
146–183. Oxford University Press.
605
Belousova, T. A., Devitsina, G. V. & Malyukina, G. A. 1983
606
Functional peculiarities of fish trigeminal system. Chem. Sen-
607
ses 8, 121–130.
608
Beukema, J. J. 1970a Angling experiments with carp (Cyprinus
609
carpio L.) II. Decreased catchability through one trial learn-
610
ing. Neth. J. Zool. 19,81–92.
611
Beukema, J. J. 1970b Acquired hook avoidance in the pike
612
Esox lucius L. fished with artificial and natural baits. J. Fish
613
Biol. 2, 155–160.
614
Bradshaw, E. L. & Bateson, P. 2000 Welfare implications of
615
culling red deer (Cervus elaphus). Anim. Welfare 9,3–24.
616
Broom, D. M. 1991 Animal welfare: concepts and measure-
617
ments. J. Anim. Sci. 69, 4167–4175.
618
Coggeshall, R. E., Leonard, R. B., Applebaum, M. L. & Willis,
619
W. D. 1978 Organization of peripheral nerves and spinal
620
roots of the Atlantic stingray, Dasyatis sabina. J. Neurophy-
621
siol. 41,97–107.
622
Dawkins, M. S. 1998 Evolution and animal welfare. Q. Rev.
623
Biol. 73, 305–328.

123
1
Nociception in fishes L. U. Sneddon and others 02PB1068.7
2
3
624
Ehrensing, R. H., Michell, G. F. & Kastin, A. J. 1982 Similar
625
antagonism of morphine analgesia by MIF-1 and naxolone
626
in Carassius auratus. Pharm. Biochem. Behav. 17, 757–761.
627
Gentle, M. J. 1992 Pain in birds. Anim. Welfare 1, 235–247.
628
Gentle, M. J. 1997 Sodium urate arthritis: effects on the sen-
629
sory properties of articular afferents in the chicken. Pain 70,
630
245–251.
631
Gentle, M. J. & Tilston, V. L. 2000 Nociceptors in the legs
632
of poultry: implications for potential pain in pre-slaughter
633
shackling. Anim. Welfare 9, 227–236.
634
Gonyou, H. W. 1994 Why the study of animal behaviour is
635
associated with the animal welfare issue. J. Anim. Sci. 72,
636
2171–2177.
637
Hallin, R. G., Torebjo
¨
rk, H. E. & Wiesenfeld, Z. 1981
638
Nociceptors and warm receptors innervated by C fibres in
639
human skin. J. Neurol. Neurosurg. Psychiatry 44, 313–319.
640
Hamamoto, D. T., Forkey, M. W., Davis, W. L., Kajander,
641
K. C. & Simone, D. A. 2000 The role of pH and osmolarity
642
in evoking the acetic acid-induced wiping response in a
643
model of nociception in frogs. Brain Res. 862, 217–229.
644
Handwerker, H. O., Anton, F. & Reeh, P. W. 1987 Discharge
645
patterns of different cutaneous nerve fibres from the rat’s
646
tail during prolonged noxious mechanical stimulation. Exp.
647
Brain Res. 65, 493–504.
648
IASP (International Association for the Study of Pain) 1979
649
쐌쐌6쐌쐌. Pain 6, 249–252.
650
Kato, Y., Kowalski, C. J. & Stohler, C. S. 2001 Habituation
651
of the early pain-specific respiratory response in sustained
652
pain. Pain 91,57–63.
653
Kavaliers, M. 1988 Evolutionary and comparative aspects of
654
nociception. Brain Res. Bull. 21, 923–931.
655
Kenshalo, D. R., Anton, F. & Dubner, R. 1989 The detection
656
and perceived intensity of noxious thermal stimuli in monk-
657
eys and in humans. J. Neurophysiol. 62, 429–436.
658
Kotrschal, K. 2000 Taste(s) and olfaction(s) in fish: a review
659
of specialized sub-systems and central integration. Eur. J.
660
Physiol. 439(Suppl.), R178–R180. 쐌쐌7쐌쐌.
661
Laitinen, M. & Valtonen, T. 1994 Cardiovascular, ventilatory
662
and total activity responses of brown trout to handling stress.
663
J. Fish Biol. 45, 933–942.
664
Lariviere, W. R. & Melzack, R. 1996 The bee venom test: a
665
new tonic-pain test. Pain 66, 271–277.
666
Leonard, R. B. 1985 Primary afferent receptive field properties
667
and neurotransmitter candidates in a vertebrate lacking
668
unmyelinated fibres. Prog. Clin. Biol. Res. 176, 135–145.
669
Lyfenko, A., Vlachova
´
, V., Vyklicky
´
, L., Dittert, I., Kress,
670
M. & Reeh, P. W. 2002 The effects of excessive heat on
671
heat-activated membrane currents in cultured dorsal root
672
ganglia neurons from neonatal rat. Pain 95, 207–214.
673
Lynn, B. 1994 The fibre composition of cutaneous nerves and
674
the classification and response properties of cutaneous affer-
1
2
Proc. R. Soc. Lond. B
3
1
PROCB: proceedings of the royal society2 26-03-03 12:23:06 Rev 16.04x PROCBC068P
3
675
ents, with particular reference to nociception. Pain Rev. 1,
676
172–183.
677
Martinez, V., Thakur, S., Mogil, J. S., Tache, Y. & Mayer,
678
E. A. 1999 Differential effects of chemical and mechanical
679
colonic irritation on behavioural pain response to intraperi-
680
toneal acetic acid in mice. Pain 81, 179–186.
681
Mason, G. J., Cooper, J. & Clarebrough, C. 2001 Frustrations
682
of fur-farmed mink: mink may thrive in captivity but they
683
miss having water to romp about in. Nature 410,35–36.
684
Matthews, G. & Wickelgren, W. O. 1978 Trigeminal sensory
685
neurons of the sea lamprey. J. Comp. Physiol. A 123, 329–
686
333.
687
Matzner, O. & Devor, M. 1987 Contrasting thermal sensitivity
688
of spontaneously active A- and C fibres in experimental
689
nerve end neuromas. Pain 30, 373–384.
690
Molony, V., Kent, J. E. & McKendrick, I. J. 2002 Validation
691
of a method for assessment of acute pain in lambs. Appl.
692
Anim. Behav. Sci. 76, 215–238.
693
Roughan, J. V. & Flecknell, P. A. 2001 Behavioural effects of
694
laparotomy and analgesic effects of ketoprofen and carprofen
695
in rats. Pain 90,65–74.
696
Roveroni, R. C., Parada, C. A., Cecilia, M., Veiga, F. A. &
697
Tambeli, C. H. 2001 Development of a behavioural model
698
of TMJ pain in rats: the TMJ formalin test. Pain 94, 185–
699
191.
700
Sneddon, L. U. 2002 Anatomical and electrophysiological
701
analysis of the trigeminal nerve in the rainbow trout,
702
Oncorhynchus mykiss. Neurosci. Lett. 319, 167–171.
703
Sneddon, L. U. 2003 Trigeminal somatosensory innervation of
704
the head of a teleost fish. Brain Res. (In the press.)쐌쐌8쐌쐌
705
Snow, P. J., Plenderleith, M. B. & Wright, L. L. 1993 Quanti-
706
tative study of primary sensory neurone populations of three
707
species of elasmobranch fish. J. Comp. Neurol. 334,97–103.
708
Stevens, C. W. 1992 Alternatives to the use of mammals for
709
pain research. Life Sci. 50, 901–912.
710
Torebjo
¨
rk, H. E. & Hallin, R. G. 1974 Identification of affer-
711
ent C units in intact human skin nerves. Brain Res. 67,
712
387–403.
713
Walters, E. T. 1996 Comparative and evolutionary aspects of
714
nociceptor function. In Neurobiology of nociceptors (ed. C.
715
Belmonte & F. Cervero), pp. 92–116. Oxford University
716
Press.
717
Yeomans, D. C. & Proudfit, H. K. 1996 Nociceptive responses
718
to high and low rates of noxious cutaneous heating are
719
mediated by different nociceptors in the rat: electrophysiol-
720
ogical evidence. Pain 68, 141–150.
721
Zimmerman, M. 1986 Physiological mechanisms of pain and
722
its treatment. Klinische Ana
¨
esthesiol. Intensivtherapie 32,1–19.
723
724
As this paper exceeds the maximum length normally permitted, the
725authors have agreed to contribute to production costs. 726