Evoked potentials in the Atlantic cod following pu

Authors: Stian Ludvigsen Niels C Stenklev Helge K Johnsen Einar Laukli Dagfinn Matre Øyvind AasHansen

Publish Date: 2013/07/30

Volume: 40, Issue: 1, Pages: 173-181

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Abstract

Aspects of peripheral and central nociception have previously been studied through recording of somatosensory evoked potentials SEPs to putative noxious stimuli in specific brain regions in a few freshwater fish species In the present study we describe a novel minimally invasive method for recording SEPs from the central nervous system of the Atlantic cod Gadus morhua Cutaneous electric stimulation of the tail in 15 fish elicited SEPs at all stimulus intensities 2 5 10 and 20 mA with quantitative properties corresponding to stimulus intensity In contrast to previous fish studies the methodological approach used in Atlantic cod in the current study uncovered a number of additional responses that could originate from multiple brain regions Several of these responses were specific to stimulation at the highest stimulus intensities possibly representing qualitative differences in central processing between somatosensory and nociceptive stimuliThe question of nociception and a possible capacity for pain perception in fish represents a topical and highly controversial issue Braithwaite and Huntingford 2004 Chandroo et al 2004 Huntingford et al 2006 Rose 2002 Rose 2007 Rose et al 2012 Sneddon 2011 In humans pain perception consists of two associated but distinct components nociception and pain Loeser and Treede 2008 The former is prevalent in the animal kingdom and concerns the ability to detect harmful noxious stimuli which requires an appropriate sensory apparatus Kavaliers 1988 Smith and Lewin 2009 Pain however includes not only a sensory component but is also a psychological state which includes an unpleasant emotional experience IASP 1979 Loeser and Treede 2008 Merskey et al 1994 Whereas nociceptive processing may occur unconsciously in both lower and higher regions of the central nervous system CNS pain perception requires mental awareness consciousness which presuppose a highly developed brain Brooks and Tracey 2005 Treede et al 1999Given the impossibility of asking an animal whether it feels pain one criterion put forward when assessing nociception and the potential for pain perception in animals is that there has to be a pathway from nociceptors in the periphery to higher brain regions Bateson 1992 Dunstan et al 1991 One way to map such a pathway is to record somatosensory evoked potentials SEPs SEPs are weak electric responses in the CNS following stimulation of peripheral sensory nerves Evaluation of SEPs is an important tool in research on nociception and pain in mammals Kakigi et al 2000 2005 In a few species of freshwater fishes goldfish Carassius auratus rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar it has previously been demonstrated that putatively nonnoxious and noxious stimulation elicited SEPs in different brain regions including the telencephalon Dunlop and Laming 2005 Nordgreen et al 2007To proceed further in the debate on nociception and potential pain perception in fish more knowledge on central nociceptive processing is needed In the present study the main aim was to present a novel minimally invasive approach to assess evoked potentials with putatively nociceptive stimuli in fish and also to investigate whether we could reproduce findings in freshwater species on a marine teleost fish the Atlantic cod Gadus morhua Our minimally invasive method was adapted from studies of auditory evoked potentials in fish Faucher et al 2009 Kenyon et al 1998 and from studies of evoked potentials with nociception and potential pain in human infants eg Slater et al 2010a b c Briefly this method involves temporal summation of repetitive stimuluslocked recordings but rather than using intracranial electrodes in spatially designated brain areas the evoked potentials are recorded from the EEG of the whole brain by using subcutaneous electrodesFifteen artificially reared Atlantic cod G morhua measuring 3038 cm were used in these experiments The fish were transported from the Tromsø Aquaculture Research Station in Kårvik to the University of Tromsø and held in large aerated seawater holding tanks connected to a flowthrough system water temperature 7 °C at least 5 days before initiation of experiments The fish were not fed during this time Experiments were performed in accordance with the guidelines of the Norwegian Animal Welfare Act National Animal Research Authority of Norway application approval number 11462011 which adheres to the European Convention for the Protection of Vertebrate Animals used for Experimentation and other Scientific Purposes Council of Europe 1998The custombuilt fish cradle consisted of a PVCpipe cut in half and tilted at an angle so that the seawater/anesthetic could exit the cradle by gravitation flow direction indicated by dotted arrows S supports bracket that kept the fish fairly level and ensured that the tail and the stimulating electrodes denoted by lightning bolt and arrow were kept dry Positioning of the recording electrodes is indicated on top of the head Figure also shows frontal view the cradleInsulated stainless steel recording electrodes Biologic Systems Corp Mundelein IL USA with only the tip exposed length 5 mm diameter 1 mm were placed subcutaneously between the eyes along the midsagittal plane of the fish so that the recording electrodes were positioned above the telencephalon The two electrodes were positioned in the midline 10 mm apart while the ground electrode was positioned close but slightly anterior to the foremost recording electrode The signal was amplified ×100000 using a CP122 AC/DC strain gage amplifier Grass Instrument Co Warwick RI USA and filtered through a 50Hz bandstop filter with high pass 1 Hz and low pass 1000 Hz before being digitized sampling frequency 1600 Hz and fed into a computer AEP software version 610 Biologic Systems Corp Mundelein IL USA was used for acquisition and analysis of data Impedance was measured before each experiment and was always below 3 kΩTwo blunt stainless steel electrodes glued to a strip of Velcro 35 mm apart were used for cutaneous stimulation The Velcro was attached around the base of the tail with the two electrodes positioned laterally so that one was above the lateral line on the right side and the other was below the lateral line on the left side Care was taken to avoid contact of the electrodes with the lateral line The distance between stimulating and recording electrodes was on average 192 cm Stimulus trains consisting of 30 rectangular electrical pulses with a duration of 1 ms and an interstimulus interval of 3 s were given by a custombuilt stimulator at each intensity 2 5 10 and 20 mA to all animals The stimulator was triggered by a Navigator®PRO Biologic Systems Corp Mundelein IL USA preamplifier controlled by the AEP software Responses were recorded in a 320ms time window from the stimulus onset with the duration of the recordings and the number of stimuli being set from preceding pilot experiments For the pilot experiments the time window used was 01000 ms but this was shortened to 320 ms for subsequent recordings as there were no responses beyond that During the pilot phase of the study postmortem stimulation with accompanying recordings was performed to exclude the presence of electromagnetic artifacts The fish were immediately euthanized after the experiment with a blow to the head

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