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

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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.
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Received 5 December 2002
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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).
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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 animals 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 animals 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
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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 (0.115.0 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 (30100 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) saline0.1 ml sterile
279 saline injected (25 g needle and 1 ml syringe) into frontal lips;
280 (ii) venom0.1 ml bee venom (1 mg ml
1
sterile saline) injected
281 into frontal lips; (iii) acid0.1 ml acetic acid (0.1% in sterile
282 saline) injected into frontal lips; and (iv) controlfish 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 MannWhitney 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 MannWhitney 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
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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-
321kalWallis 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
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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
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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-
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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
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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
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... e.g., (2); see also (3)(4)(5)(6)(7)]. Today, thanks in part to the trailblazing work of Dr. Victoria Braithwaite celebrated in this Special Topic collection, there is no disputing what an important issue this is, and also no argument as to whether bony fishes possess functioning nociceptors [e.g., rainbow trout (Oncorhynchus mykiss): (8,9), goldfish (Carassius auratus): (10), common carp (Cyprinius carpio): (11)]. But the question of whether fish are aware of noxious stimuli, and feel true pain, remains contested and controversial, stimulating considerable debate. ...
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Debates around fishes' ability to feel pain concern sentience : do reactions to tissue damage indicate evaluative consciousness (conscious affect), or mere nociception? Thanks to Braithwaite's discovery of trout nociceptors, and concerns that current practices could compromise welfare in countless fish, this issue's importance is beyond dispute. However, nociceptors are merely necessary, not sufficient, for true pain, and many measures held to indicate sentience have the same problem. The question of whether fish feel pain – or indeed anything at all – therefore stimulates sometimes polarized debate. Here, we try to bridge the divide. After reviewing key consciousness concepts, we identify “red herring” measures that should not be used to infer sentience because also present in non-sentient organisms, notably those lacking nervous systems, like plants and protozoa (P); spines disconnected from brains (S); decerebrate mammals and birds (D); and humans in unaware states (U). These “S.P.U.D. subjects” can show approach/withdrawal; react with apparent emotion; change their reactivity with food deprivation or analgesia; discriminate between stimuli; display Pavlovian learning, including some forms of trace conditioning; and even learn simple instrumental responses. Consequently, none of these responses are good indicators of sentience. Potentially more valid are aspects of working memory, operant conditioning, the self-report of state, and forms of higher order cognition. We suggest new experiments on humans to test these hypotheses, as well as modifications to tests for “mental time travel” and self-awareness (e.g., mirror self-recognition) that could allow these to now probe sentience (since currently they reflect perceptual rather than evaluative, affective aspects of consciousness). Because “bullet-proof” neurological and behavioral indicators of sentience are thus still lacking, agnosticism about fish sentience remains widespread. To end, we address how to balance such doubts with welfare protection, discussing concerns raised by key skeptics in this debate. Overall, we celebrate the rigorous evidential standards required by those unconvinced that fish are sentient; laud the compassion and ethical rigor shown by those advocating for welfare protections; and seek to show how precautionary principles still support protecting fish from physical harm.
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... Studies using rainbow trout Oncorhynchus mykiss have demonstrated the existence of a nociceptive system like those of other vertebrates, with receptors in the skin and two types of trigeminal nerve fibres that carry nociceptive information to the brain. Such a conduction of information to the brain generates the perception of pain (Sneddon et al., 2003). ...
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Menthol has been recommended for fish anaesthesia in recent studies. However, no observations of brain electrical activity, electromyography and electrocardiogram have been reported to date in in vivo experiments using menthol. In order to fill the gaps at the electrophysiological and behavioural levels, juveniles of tambaqui, Colossoma macropomum were used as live models and subjected to two experimental conditions: I - Behavioural assessment using menthol at 100 mg.L⁻¹; II - Characterization of electroencephalography (EEG), electromyography (EMG), electrocardiography (ECG), and opercular beat rate (OBR) using menthol at 100 mg.L⁻¹. The fish were assayed into three groups for all characterizations: (a) control; (b) ethanol (vehicle control); and (c) menthol at100 mg.L⁻¹, nine animals per group were used for each marker, one fish being considered a replicate (n = 9) and used only once. On the EEG, it was observed that menthol induced decreased mean power in cerebral activity, indicating the deepening of the anaesthesia, showing that menthol led to reversible CNS depression, with the resumption of mean power after washout, devoid of seizures or any other observable alterations. The EMG showed a decreased skeletal muscle tone, with no excitability or muscle spasms. The ECG showed a reduced heart rate, however, menthol did not elicit important alterations during induction or recovery in the duration of the QRS complex (ventricular depolarization). For all records the sinus rhythm was maintained, menthol did not lead to deleterious QT interval (ventricular contraction) prolongation, and the RR interval (time between two successive QRS complexes) corroborated a reversible anaesthetic effect. OBR showed the maintenance of the ventilatory rate during induction and recovery, which indicates a reduced risk of causing severe hypoxia. Overall, the electrophysiological data reinforced the results observed in the behavioural assessment. The findings indicate that menthol proved to be an effective and safe anaesthetic for tambaqui juveniles, as it did not cause major changes in the pattern of tracings, and therefore, should be considered a standard chemical compound of plant origin for the purpose of full immobilization and general anaesthesia of C. macropomum.
... In contrast to the original proposal that (teleost) fish lack the prerequisite neural architecture for phenomenal consciousness including pain, and thus cannot feel pain (Key, 2015), studies have refuted this statement at the molecular (Sneddon, 2019), behavioral (Sneddon et al., 2003;Braithwaite and Boulcott, 2007) and circuital/anatomical level (Maximino et al., 2013). Furthermore, recent results showed that the cleaner wrasse FIGURE 1 | The connections from the peripheral olfactory epithelia to the olfactory bulbs are highly conserved in vertebrates. ...
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Within the forebrain the olfactory sensory system is unique from other sensory systems both in the projections of the olfactory tract and the ongoing neurogenic potential, characteristics conserved across vertebrates. Olfaction plays a crucial role in behaviors such as mate choice, food selection, homing, escape from predators, among others. The olfactory forebrain is intimately associated with the limbic system, the region of the brain involved in learning, memory, and emotions through interactions with the endocrine system and the autonomic nervous system. Previously thought to lack a limbic system, we now know that teleost fishes process emotions, have exceptional memories, and readily learn, behaviors that are often associated with olfactory cues. The association of neuromodulatory hormones, and more recently, the immune system, with odor cues underlies behaviors essential for maintenance and adaptation within natural ecological niches. Increasingly anthropogenic perturbations affecting ecosystems are impacting teleost fishes worldwide. Here we examine the role of the olfactory tract as the neural basis for the integration of environmental cues and resulting behaviors necessary for the regulation of biotic interactions that allow for future adaptation as the climate spins out of control.
... Here the requirement for a good welfare is that the animal should feel well and be free from negative experiences such as pain or fear. Studies have shown and demonstrated the presence of nociceptors involved in pain feeling mechanisms in fish (Ashley et al., 2007;Sneddon et al., 2003), which shows that pain is possible and should therefore be avoided. The nature-based definition suggests that each animal has an inherent biological nature that must be expressed. ...
Article
Behavioural traits have been shown to have implications in fish welfare and growth performances in aquaculture. If several studies have demonstrated the existence of repeatable and heritable behavioural traits (i.e., animal personality), the methodology to assess personality in fishes is often carried out in solitary context, which appears to somewhat limit their use from a selective breeding perspective because these tests are too time consuming. To address this drawback, group-based tests have been developed. In Nordic country, Arctic charr (Salvelinus alpinus) is widely used in aquaculture, but no selection effort on behavioural traits has yet been carried out. Specifically, in this study we examined if risk-taking behaviour was repeatable and correlated in group and solitary context and if the early influences of physical environment affect the among-individual variation of behavioural trait across time in order to verify whether a group risk-taking test could be used as a selective breeding tool. Here, we found that in both contexts and treatments, the risk-taking behaviour was repeatable across a short period of 7 days. However, no cross-context consistency was found between group and solitary, which indicates that individual Arctic charr express different behavioural trait in group and solitary.
... For example, work by Victoria Braithwaite on simple manipulations in the environment and feeding of juvenile cod (Gadus morhua) (2) opened my eyes to the possibility that fish might be capable of more complex and rewarding lives than I had previously thought possible, but this did not significantly affect my actions. In contrast, discussions on pain in fish based on studies conceived by Victoria Braithwaite and Mike Gentles (3,4) convinced me that fish should be given the benefit of the doubt with regard to pain. As a result, I have changed my behavior, with the prevention, or alleviation of (potential) pain in fish becoming a personal priority. ...
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As a veterinarian and academic in aquaculture, in my personal experience, most farmers are concerned for their animals and want to take good care of them. There has been substantial improvement in the welfare of farmed fish in recent decades, but improvements have been inconsistent across culture systems and species. Where there has been a lack of progress, it is not simply due to the more obvious barriers, for example, lack of clear messages, lack of effective dissemination, or cost of implementation. Why have the good intentions of farmers and research by academics failed to improve the care of many farmed fish? The reasons would appear to be complex; however, human behavioral theory (this term is used to differentiate from animal ethology) offers both a conceptual framework and practical guidelines for improving the care of fish by influencing the behavior of farmers. Here, I present some background context and apply human behavioral theory to examples of on-farm care of fish.
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‘My fish and I’ is an account of the diversity of human-fish interactions. This includes their benefits, detriments/harms as well as their moral and animal welfare. Fish are not easily perceived as individual animals having mental states, interests, needs and a degree of individuality. Additionally, fish have been handled as a simple resource in innumerable human interactions. Important ethical approaches address animal-human interactions based upon the individual’s cognitive ability and capacity to feel pleasure and pain. Given the ample evidence that fish have neuroanatomical structures that support the capacity to feel (sentience) and have complex behavioural and cognitive abilities, a moral duty is imposed upon us. Some human-centered and eco-centered moral views complement different perceptions of the nature of our relationship with fish. This occurs both at the individual level and as species or populations face a serious need for conservation. The concepts and assessments in the developments of animal welfare science provide ample basis for an evolution in the quality of human-fish interactions. However, many stakeholders must take part in this evolution. This is especially true as it concerns those areas of activity involving many individual fish and higher levels of suffering. Examples of these are aquaculture and commercial fisheries where there is much more at stake. Consumers will have the last word in this role, namely by reducing fish consumption.
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The exponential rise of the zebrafish (Danio rerio) as a model organism in biomedical research has far outstripped our understanding of basic husbandry and welfare for this species. As a case in point, here we investigate the efficacy and welfare impact of different euthanasia methods for zebrafish. Not only is a humane death central to welfare and the 3Rs, but stress during euthanasia can change scientific outcomes. However, the most frequently used methods of euthanasia have multiple shortcomings with regard to animal welfare and human safety. In this study, we propose the use of propofol for immersion euthanasia of adult zebrafish. Propofol has been known to rapidly induce anesthesia in many species, including zebrafish, but its efficacy as a euthanasia agent for zebrafish has not fully been explored. In this study, adult zebrafish were euthanized by immersion on one of 5 different preparations: ice bath, 250 ppm MS222, 600 ppm lidocaine hydrochloride, 100 ppm propofol, or 150 ppm propofol for 20 or 30 min. Display of aversive behaviors, time to loss of righting reflex, time to cessation of opercular movement, and time to recovery after transfer to clean tank water were assessed and recorded. Propofol at both concentrations induced loss of righting reflex and loss of opercular movement more quickly than did MS222 or lidocaine hydrochloride and caused no display of aversive behaviors as seen with ice bath or lidocaine exposure. However, fish exposed to propofol at either concentration for 20 min sometimes recovered, whereas a 30-min exposure was sufficient for euthanasia of all fish tested. These findings suggest that exposure to propofol for a duration of at least 30 min quickly and effectively euthanizes adult zebrafish without compromising end of-life welfare.
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Evidence from comparative morphology and electrophysiology suggests that both, olfaction and taste in fish serve different ecological roles. The lateral olfactory system (dorsolateral olfactory bulb glomeruli and lateral olfactory tract) and the external taste buds are probably specialized for food search and amino acid discrimination. The medial olfactory system (basomedial olfactory bulb glomeruli and medial olfactory tract) and the solitary chemosensory taste cells, however, may have their roles in intra-and interspecific interactions (discriminating pheromones by olfaction, bile components by both olfaction and taste). Whereas stimulation of the taste systems alone triggers reflexes, complex, conditional or conditioned behaviours are only released when the olfactory system is intact. This points at the importance of telencephalic and diencephalic integration of olfactory and taste inputs. Consequently, caution is appropriate concerning simplistic interpretations of deprivation experiments.
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Changes in total activity, heart and ventilation rates were observed in 2-year-old brown trout, following handling stress, using non-contact bioelectronic monitoring equipment. Experiments were carried out in laboratory conditions at water temperatures below 4 degrees C. Transfer between tanks as well as 5 min restraint stress increased the total activity of fish for 24 to 48 h, after which it declined to near the pre-stress lever. The transfer and struggle both elevated the heart rate for 3 to 4 days. Ventilation rate was elevated to a maximum of about 30% above the nominal level and recovered within 3 to 4 days. Both heart and ventilation rates were higher in feeding fish relative to fasting fish after stress and rates remained higher throughout a 7 day period of recovery. A diel rhythm of lower rates during the night appeared in both heart and ventilation rates within 3 to 4 days after handling stress.