Articles in PresS. J Neurophysiol (May 21, 2014). doi:10.1152/jn.00228.2014
1
Direct activation of the Mauthner cell by electric field
2
pulses drives ultra-rapid escape responses
3 4 5
Kathryn M. Tabor1, Sadie A. Bergeron1, Eric J. Horstick1, Diana C. Jordan1,
6
Vilma Aho2, Tarja Porkka-Heiskanen2, Gal Haspel3 and Harold A. Burgess1*
7 8
1
9
Child Health and Human Development, Bethesda, MD 20892
Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of
10
2
Institute of Biomedicine, University of Helsinki, Helsinki, Finland
11
3
Department of Biological Sciences, New Jersey Institute of Technology, Newark, NJ
12
07102
13 14
Running head:
Electric field pulses activate the Mauthner cell
15 16
* Address for correspondence: Harold A. Burgess; Building 6B, Room 3B308; 6 Center
17
Drive; Bethesda, MD 20892 ; Email: [email protected] ; phone: 301-402-6018 ;
18
fax: 301-496-0243
19 20
Contributions: K.M.T., T.P-H., G.H., and H.A.B. designed the experiments. K.M.T,
21
S.A.B., E.J.H, D.C.J., V.A. and H.A.B. performed the experiments. All authors analyzed
22
results. K.M.T and H.A.B. wrote the manuscript.
23
Copyright © 2014 by the American Physiological Society.
24
Abstract
25
Rapid escape swims in fish are initiated by the Mauthner cells, giant reticulospinal
26
neurons with unique specializations for swift responses. The Mauthner cells directly
27
activate motoneurons and facilitate predator detection by integrating acoustic,
28
mechanosensory and visual stimuli. In addition, larval fish show well-coordinated escape
29
responses when exposed to electric field pulses (EFPs). Sensitization of the Mauthner cell
30
by genetic overexpression of the voltage-gated sodium channel SCN5 increased EFP
31
responsiveness, whereas Mauthner ablation using an engineered variant of nitroreductase
32
with increased activity (epNTR), eliminated the response. The reaction time to EFPs is
33
extremely short, with many responses initiated within 2 ms of the EFP. Large neurons,
34
such as Mauthner cells, show heightened sensitivity to extracellular voltage gradients.
35
We therefore tested if the rapid response to EFPs was due to direct activation of the
36
Mauthner cells, bypassing delays imposed by stimulus detection and transmission by
37
sensory cells. Consistent with this, calcium imaging indicated that EFPs robustly
38
activated the Mauthner cell, but only rarely fired other reticulospinal neurons. Further
39
supporting this idea, pharmacological blockade of synaptic transmission in zebrafish did
40
not affect Mauthner cell activity in response to EFPs. Moreover, Mauthner cells
41
transgenically expressing a tetrodotoxin (TTX) resistant voltage-gated sodium channel
42
retained responses to EFPs despite TTX suppression of action potentials in the rest of the
43
brain. We propose that EFPs directly activate Mauthner cells due to their large size,
44
thereby driving ultra-rapid escape responses in fish.
45 46
Keywords: escape response, Mauthner cell, electric pulse, nitroreductase, SCN5
47
Introduction
48 49
Teleost fish have among the fastest reaction time for escape responses in the animal
50
kingdom, rapidly accelerating away from the strike path of predators within tens of
51
milliseconds. Response latency is a critical factor in predator evasion, enabling prey to
52
escape the strike zone and freely maneuver while predators are engaged in stereotyped
53
capture movements. Escape responses in fish are initiated by the Mauthner cells, giant
54
reticulospinal neurons with unique specializations for swift responses (Faber and Korn
55
1978). Underlining their central role in the escape circuit, Mauthner cells are activated by
56
sudden stimuli in diverse sensory modalities. Mauthner cells receive monosynaptic
57
chemical and electrical input from statoacoustic, trigeminal and lateral line ganglia and
58
visual input relayed via the tectum (Faber and Korn 1978; Kimmel et al. 1990). A single
59
action potential transmitted down the large diameter Mauthner axon is sufficient to
60
activate a startle response (Nissanov et al. 1990), depolarizing motoneurons through both
61
mono and di-synaptic pathways (Fetcho 1992). The short neuronal pathway from sensory
62
input to motoneuron activation thus guarantees fast reaction times.
63 64
Low survival rates among fish larvae necessitate that escape responses emerge very early
65
in development and indeed by 5 days post fertilization (dpf), zebrafish larvae execute
66
rapid escape swims when threatened by acoustic, tactile or looming visual cues (reviewed
67
in (Fero et al. 2011)). Natural predators of zebrafish larvae include adult fish and
68
dragonfly nymphs (Engeszer et al. 2007) which use a hydraulic mechanism to achieve
69
high velocity labial strikes (Tanaka and Hisada 1980). Accordingly, behavioral response
2
70
latencies in larvae are short - 3 to 10 ms for acoustic and tactile stimuli (Kohashi and Oda
71
2008; Liu and Fetcho 1999) and around 400 ms for looming visual stimuli (Facchin et al.
72
2009). However the fastest reaction time for escape responses, frequently less than 2 ms,
73
is seen in response to an electric field pulse (EFP). Many fish species initiate well-
74
coordinated escape swims in response to EFPs (Dunlop et al. 2006; Webb 1980; 1976;
75
Yokogawa et al. 2012). Apart from their use in analyzing escape responses, EFPs are
76
widely used in behavioral assays in fish, especially in paradigms for learning and
77
memory where EFPs act as unconditioned aversive stimuli (Aoki et al. 2013; Bitterman
78
1964; Portavella et al. 2002; Rawashdeh et al. 2007; Shcherbakov et al. 2005; Valente et
79
al. 2012). In addition, EFPs have been used to assess arousal states (Yokogawa et al.
80
2007), study nociception (Dunlop et al. 2006), and analyze autonomic and behavioral
81
responses to stressors (Agetsuma et al. 2010; Lee et al. 2010; Mann et al. 2010).
82 83
Despite this extensive use in research, the sensory modality through which EFPs trigger
84
behavioral responses in fish has not been identified, although it has been assumed that, as
85
in mammals, EFPs act on cutaneous nociceptors (Dunlop et al. 2006). However, we
86
observed that in zebrafish larvae, the first trunk movements in response to an EFP are
87
detectable within 2 ms of the stimulus onset. The extreme short latency of EFP responses
88
suggested that they are mediated by an unusual neuronal mechanism. Using genetic and
89
physiological techniques we investigated the neuronal circuitry by which EFPs elicit
90
ultra-rapid escape responses.
91 92
Materials and Methods
3
93 94
Fish husbandry. Zebrafish husbandry and tol1 transgenesis has been described
95
(Yokogawa et al. 2012). Et(SCP1:Gal4ff)y264 (y264), Et(REx2-cfos:Gal4ff)y269 (y269)
96
and Et(REx2-cfos:Gal4ff)y270 (y270) were isolated in an enhancer trap screen (Bergeron
97
et al. 2012). y264 shows strong expression of Gal4 in the Mauthner cell and has
98
additional stochastic expression in other reticulospinal neurons, the anterior lateral line
99
ganglia and sparse cells in the spinal cord, hindbrain, cerebellum and forebrain (Figure
100
3B). For Tg(UAS-E1b:BGi-epNTR-TagRFPT-oPre)y268 (UAS:epNTR-RFP), zebrafish
101
codon optimized nfsB was synthesized (Genscript), fused to TagRFPT and cloned into
102
pT1UMP (Yokogawa et al. 2012). Mutations T41L, N71S and F124W (Jaberipour et al.
103
2010; LinWu et al. 2012) were introduced by PCR mutagenesis to generate an 'extra-
104
potent' nitroreductase (epNTR). Recently a similar set of mutations was independently
105
tested in zebrafish and also found to improve nitroreductase activity (Mathias et al.
106
2014). For Tg(UAS-E1b:BGi-SCN5a-v2a-TagRFPT)y266 (UAS:SCN5), human SCN5a
107
(9053152, Thermo Scientific) was fused to v2a-TagRFPT and cloned in pT1UMP. For
108
Tg(UAS-E1b:BGi-GCaMP6s-v2a-mCherry-v2a-lynCFP-oPre)y267 (UAS:GCaMP6s),
109
K78H, T381R, S383T and R392G mutations (Chen et al. 2013) were introduced into
110
zebrafish optimized GCaMP5g (Genscript). Tg(UAS:nfsB-mCherry) was made using the
111
construct reported by Davison et al.(Davison et al. 2007). Other fish larvae were fathead
112
minnows (Pimephales promelas, MBL Aquaculture), Mexican cavefish (Astyanax
113
mexicanus, kind gift of William Jeffery, University of Maryland) and medaka (Oryzias
114
latipes, kind gift of Fumihito Ono, NIAAA). In vivo experimental protocols were
115
approved by the local animal care and use committee.
4
116 117
Immunohistochemistry. Antibodies were GFP (1:1000, A-11122, Invitrogen), Kaede
118
(1:1000, PM012, MBL international), and SCN5a (1:1000, C144743, LifeSpan
119
BioSciences) and Alexa Fluor 488- and 546- conjugated secondary antibodies (1:400,
120
Invitrogen). Images were captured using a Leica TCS-SP5 II confocal microscope.
121 122
Behavioral analysis. Individual and group testing were performed in a 4.2 cm square
123
arena above a translucent diffuser illuminated with an LED array. Individual testing of
124
acoustic responses was performed in grid of 1 cm2 chambers. Responses were recorded
125
with a high speed camera (DRS Lightning RDT/1, DEL Imaging) at 1000 frames per
126
second, and analyzed with Flote software (Burgess and Granato 2007). Acoustic startle
127
responses, elicited as previously described, occur in two waves: short-latency C-starts
128
(SLC) and long-latency C-starts (Burgess and Granato 2007). Except for Figure 1C, only
129
data from SLCs was analyzed. As larvae were most responsive to EFPs when oriented
130
toward the anode, except for Figure 2A,B, results are for larvae oriented within 22.5° of
131
this direction. Stimuli were generated using a digital-to-analog card (PCI-6221; National
132
Instruments). EFPs were square DC pulses (2 ms duration, 1-9 V amplitude except as
133
otherwise noted) or sinusoidal AC pulses (0.5-2 ms period, 2 ms duration, 9 V amplitude)
134
across stainless steel wire mesh electrodes 1 or 4.2 cm apart for free-swimming larvae, or
135
2.3 cm apart for head-embedded larvae.
136 137
Calcium imaging. Calcium fluorescence was monitored either using y264 ;
138
UAS:GCaMP6s larvae or Mauthner cells retrogradely labeled by injecting Calcium
5
139
Green-1 dextran (C3713; Invitrogen) into the spinal cord. Larvae were head-embedded in
140
2% agarose with tail movement unrestricted. Fluorescence was monitored with an
141
Axioimager compound microscope (Carl Zeiss) and tail movements recorded using a
142
µEye camera (IDS Imaging). Fluorescence intensities were extracted using custom
143
software. The microscope stage was affixed with a speaker and a collar with electrodes
144
was positioned inside the dish.
145 146
Pharmacology. DL-2-Amino-5-phosphonopentanoic acid (APV, 100 µM, A5282,
147
Sigma), carbenoxolone (CBX, 1 µM, C4790, Sigma), NBQX (20 µM, N183, Sigma),
148
strychnine (50 µM, S8753, Sigma) and tetrodotoxin (1 µM, TTX, T-500, Alomone labs)
149
were dissolved in E3 medium. Picrotoxin (100 µM, P1675, Sigma) was dissolved in
150
DMSO. Larvae were embedded in agarose and drugs added to the bath solution and
151
injected in the brain ventricle (2-4 nl) using a picospritzer (PV820, World Precision
152
Instruments). Fish were tested before and 10 (TTX) or 30 (all others) minutes after drug
153
delivery. For behavior testing y264 ; UAS:SCN5 larvae, RFP expression was visually
154
confirmed at 4 dpf after anaesthetization with 0.03% tricaine (MS-222, Sigma). Larvae
155
were allowed to recover 40 h in E3 before testing.
156 157
Ablations. y264 ; UAS:epNTR-RFP, y269 ; UAS:epNTR-RFP and y269 ; UAS:nfsB-
158
mCherry larvae and RFP negative siblings (controls) were treated with 10 mM
159
metronidazole in E3 from 3-5 dpf. To detect cell death during genetic ablation, larvae
160
were immersed for 1 h in 8 μm PhiPhiLux G1D2 (OncoImmunin) and washed three times
161
before live imaging as previously described (Yokogawa et al. 2012). Laser ablations were
6
162
performed in Gt(T2KSAG)j1229a (J1229) larvae which express GFP in the Mauthner cell
163
(Burgess et al. 2009). Mauthner cells, or RoV3 neurons for controls, were ablated using a
164
Leica TCS-SP5 II 2-photon confocal microscope with a 20x 0.95 NA objective and an
165
800 nm laser. Immunolabeling against GFP confirmed successful ablation of 70% of
166
targeted cells. Only data from confirmed ablations was analyzed.
167 168
Statistical analysis. Analyses were performed using Gnumeric
169
(http://projects.gnome.org/gnumeric/). Data in figures and text are mean and s.e.m.
170 171
Results
172 173
EFPs induce well-coordinated swimming movements in larval zebrafish which strongly
174
resemble C-start escape responses triggered by acoustic stimuli (Figure 1A,B,
175
supplemental movie 1). Larvae show two types of responses to acoustic stimuli
176
distinguished by latency and kinematics (Burgess and Granato 2007). In contrast the vast
177
majority of EFP responses were initiated within 10 ms of the stimulus (Figure 1C). Since
178
both AC and DC EFPs have been used in behavioral assays in fish we tested the
179
behavioral responsiveness to both stimulus types. Responsiveness to EFPs was greater for
180
square (DC) field pulses compared to sinusoidal (AC) pulses of the same duration and
181
amplitude (Figure 1D). Using long duration field pulses (10 ms) we observed that most
182
EFP responses were evoked by the onset of the positive field potential, not the pulse
183
offset (Figure 1E), and increasing the duration of the square pulse did not increase
184
responsiveness (Figure 1F). Surprisingly larvae showed a faster reaction time to EFPs
7
185
than to acoustic stimuli (Figure 1C inset, electric, 2.7±0.06 ms; acoustic, 5.1±0.07 ms,
186
p
1
Direct activation of the Mauthner cell by electric field
2
pulses drives ultra-rapid escape responses
3 4 5
Kathryn M. Tabor1, Sadie A. Bergeron1, Eric J. Horstick1, Diana C. Jordan1,
6
Vilma Aho2, Tarja Porkka-Heiskanen2, Gal Haspel3 and Harold A. Burgess1*
7 8
1
9
Child Health and Human Development, Bethesda, MD 20892
Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of
10
2
Institute of Biomedicine, University of Helsinki, Helsinki, Finland
11
3
Department of Biological Sciences, New Jersey Institute of Technology, Newark, NJ
12
07102
13 14
Running head:
Electric field pulses activate the Mauthner cell
15 16
* Address for correspondence: Harold A. Burgess; Building 6B, Room 3B308; 6 Center
17
Drive; Bethesda, MD 20892 ; Email: [email protected] ; phone: 301-402-6018 ;
18
fax: 301-496-0243
19 20
Contributions: K.M.T., T.P-H., G.H., and H.A.B. designed the experiments. K.M.T,
21
S.A.B., E.J.H, D.C.J., V.A. and H.A.B. performed the experiments. All authors analyzed
22
results. K.M.T and H.A.B. wrote the manuscript.
23
Copyright © 2014 by the American Physiological Society.
24
Abstract
25
Rapid escape swims in fish are initiated by the Mauthner cells, giant reticulospinal
26
neurons with unique specializations for swift responses. The Mauthner cells directly
27
activate motoneurons and facilitate predator detection by integrating acoustic,
28
mechanosensory and visual stimuli. In addition, larval fish show well-coordinated escape
29
responses when exposed to electric field pulses (EFPs). Sensitization of the Mauthner cell
30
by genetic overexpression of the voltage-gated sodium channel SCN5 increased EFP
31
responsiveness, whereas Mauthner ablation using an engineered variant of nitroreductase
32
with increased activity (epNTR), eliminated the response. The reaction time to EFPs is
33
extremely short, with many responses initiated within 2 ms of the EFP. Large neurons,
34
such as Mauthner cells, show heightened sensitivity to extracellular voltage gradients.
35
We therefore tested if the rapid response to EFPs was due to direct activation of the
36
Mauthner cells, bypassing delays imposed by stimulus detection and transmission by
37
sensory cells. Consistent with this, calcium imaging indicated that EFPs robustly
38
activated the Mauthner cell, but only rarely fired other reticulospinal neurons. Further
39
supporting this idea, pharmacological blockade of synaptic transmission in zebrafish did
40
not affect Mauthner cell activity in response to EFPs. Moreover, Mauthner cells
41
transgenically expressing a tetrodotoxin (TTX) resistant voltage-gated sodium channel
42
retained responses to EFPs despite TTX suppression of action potentials in the rest of the
43
brain. We propose that EFPs directly activate Mauthner cells due to their large size,
44
thereby driving ultra-rapid escape responses in fish.
45 46
Keywords: escape response, Mauthner cell, electric pulse, nitroreductase, SCN5
47
Introduction
48 49
Teleost fish have among the fastest reaction time for escape responses in the animal
50
kingdom, rapidly accelerating away from the strike path of predators within tens of
51
milliseconds. Response latency is a critical factor in predator evasion, enabling prey to
52
escape the strike zone and freely maneuver while predators are engaged in stereotyped
53
capture movements. Escape responses in fish are initiated by the Mauthner cells, giant
54
reticulospinal neurons with unique specializations for swift responses (Faber and Korn
55
1978). Underlining their central role in the escape circuit, Mauthner cells are activated by
56
sudden stimuli in diverse sensory modalities. Mauthner cells receive monosynaptic
57
chemical and electrical input from statoacoustic, trigeminal and lateral line ganglia and
58
visual input relayed via the tectum (Faber and Korn 1978; Kimmel et al. 1990). A single
59
action potential transmitted down the large diameter Mauthner axon is sufficient to
60
activate a startle response (Nissanov et al. 1990), depolarizing motoneurons through both
61
mono and di-synaptic pathways (Fetcho 1992). The short neuronal pathway from sensory
62
input to motoneuron activation thus guarantees fast reaction times.
63 64
Low survival rates among fish larvae necessitate that escape responses emerge very early
65
in development and indeed by 5 days post fertilization (dpf), zebrafish larvae execute
66
rapid escape swims when threatened by acoustic, tactile or looming visual cues (reviewed
67
in (Fero et al. 2011)). Natural predators of zebrafish larvae include adult fish and
68
dragonfly nymphs (Engeszer et al. 2007) which use a hydraulic mechanism to achieve
69
high velocity labial strikes (Tanaka and Hisada 1980). Accordingly, behavioral response
2
70
latencies in larvae are short - 3 to 10 ms for acoustic and tactile stimuli (Kohashi and Oda
71
2008; Liu and Fetcho 1999) and around 400 ms for looming visual stimuli (Facchin et al.
72
2009). However the fastest reaction time for escape responses, frequently less than 2 ms,
73
is seen in response to an electric field pulse (EFP). Many fish species initiate well-
74
coordinated escape swims in response to EFPs (Dunlop et al. 2006; Webb 1980; 1976;
75
Yokogawa et al. 2012). Apart from their use in analyzing escape responses, EFPs are
76
widely used in behavioral assays in fish, especially in paradigms for learning and
77
memory where EFPs act as unconditioned aversive stimuli (Aoki et al. 2013; Bitterman
78
1964; Portavella et al. 2002; Rawashdeh et al. 2007; Shcherbakov et al. 2005; Valente et
79
al. 2012). In addition, EFPs have been used to assess arousal states (Yokogawa et al.
80
2007), study nociception (Dunlop et al. 2006), and analyze autonomic and behavioral
81
responses to stressors (Agetsuma et al. 2010; Lee et al. 2010; Mann et al. 2010).
82 83
Despite this extensive use in research, the sensory modality through which EFPs trigger
84
behavioral responses in fish has not been identified, although it has been assumed that, as
85
in mammals, EFPs act on cutaneous nociceptors (Dunlop et al. 2006). However, we
86
observed that in zebrafish larvae, the first trunk movements in response to an EFP are
87
detectable within 2 ms of the stimulus onset. The extreme short latency of EFP responses
88
suggested that they are mediated by an unusual neuronal mechanism. Using genetic and
89
physiological techniques we investigated the neuronal circuitry by which EFPs elicit
90
ultra-rapid escape responses.
91 92
Materials and Methods
3
93 94
Fish husbandry. Zebrafish husbandry and tol1 transgenesis has been described
95
(Yokogawa et al. 2012). Et(SCP1:Gal4ff)y264 (y264), Et(REx2-cfos:Gal4ff)y269 (y269)
96
and Et(REx2-cfos:Gal4ff)y270 (y270) were isolated in an enhancer trap screen (Bergeron
97
et al. 2012). y264 shows strong expression of Gal4 in the Mauthner cell and has
98
additional stochastic expression in other reticulospinal neurons, the anterior lateral line
99
ganglia and sparse cells in the spinal cord, hindbrain, cerebellum and forebrain (Figure
100
3B). For Tg(UAS-E1b:BGi-epNTR-TagRFPT-oPre)y268 (UAS:epNTR-RFP), zebrafish
101
codon optimized nfsB was synthesized (Genscript), fused to TagRFPT and cloned into
102
pT1UMP (Yokogawa et al. 2012). Mutations T41L, N71S and F124W (Jaberipour et al.
103
2010; LinWu et al. 2012) were introduced by PCR mutagenesis to generate an 'extra-
104
potent' nitroreductase (epNTR). Recently a similar set of mutations was independently
105
tested in zebrafish and also found to improve nitroreductase activity (Mathias et al.
106
2014). For Tg(UAS-E1b:BGi-SCN5a-v2a-TagRFPT)y266 (UAS:SCN5), human SCN5a
107
(9053152, Thermo Scientific) was fused to v2a-TagRFPT and cloned in pT1UMP. For
108
Tg(UAS-E1b:BGi-GCaMP6s-v2a-mCherry-v2a-lynCFP-oPre)y267 (UAS:GCaMP6s),
109
K78H, T381R, S383T and R392G mutations (Chen et al. 2013) were introduced into
110
zebrafish optimized GCaMP5g (Genscript). Tg(UAS:nfsB-mCherry) was made using the
111
construct reported by Davison et al.(Davison et al. 2007). Other fish larvae were fathead
112
minnows (Pimephales promelas, MBL Aquaculture), Mexican cavefish (Astyanax
113
mexicanus, kind gift of William Jeffery, University of Maryland) and medaka (Oryzias
114
latipes, kind gift of Fumihito Ono, NIAAA). In vivo experimental protocols were
115
approved by the local animal care and use committee.
4
116 117
Immunohistochemistry. Antibodies were GFP (1:1000, A-11122, Invitrogen), Kaede
118
(1:1000, PM012, MBL international), and SCN5a (1:1000, C144743, LifeSpan
119
BioSciences) and Alexa Fluor 488- and 546- conjugated secondary antibodies (1:400,
120
Invitrogen). Images were captured using a Leica TCS-SP5 II confocal microscope.
121 122
Behavioral analysis. Individual and group testing were performed in a 4.2 cm square
123
arena above a translucent diffuser illuminated with an LED array. Individual testing of
124
acoustic responses was performed in grid of 1 cm2 chambers. Responses were recorded
125
with a high speed camera (DRS Lightning RDT/1, DEL Imaging) at 1000 frames per
126
second, and analyzed with Flote software (Burgess and Granato 2007). Acoustic startle
127
responses, elicited as previously described, occur in two waves: short-latency C-starts
128
(SLC) and long-latency C-starts (Burgess and Granato 2007). Except for Figure 1C, only
129
data from SLCs was analyzed. As larvae were most responsive to EFPs when oriented
130
toward the anode, except for Figure 2A,B, results are for larvae oriented within 22.5° of
131
this direction. Stimuli were generated using a digital-to-analog card (PCI-6221; National
132
Instruments). EFPs were square DC pulses (2 ms duration, 1-9 V amplitude except as
133
otherwise noted) or sinusoidal AC pulses (0.5-2 ms period, 2 ms duration, 9 V amplitude)
134
across stainless steel wire mesh electrodes 1 or 4.2 cm apart for free-swimming larvae, or
135
2.3 cm apart for head-embedded larvae.
136 137
Calcium imaging. Calcium fluorescence was monitored either using y264 ;
138
UAS:GCaMP6s larvae or Mauthner cells retrogradely labeled by injecting Calcium
5
139
Green-1 dextran (C3713; Invitrogen) into the spinal cord. Larvae were head-embedded in
140
2% agarose with tail movement unrestricted. Fluorescence was monitored with an
141
Axioimager compound microscope (Carl Zeiss) and tail movements recorded using a
142
µEye camera (IDS Imaging). Fluorescence intensities were extracted using custom
143
software. The microscope stage was affixed with a speaker and a collar with electrodes
144
was positioned inside the dish.
145 146
Pharmacology. DL-2-Amino-5-phosphonopentanoic acid (APV, 100 µM, A5282,
147
Sigma), carbenoxolone (CBX, 1 µM, C4790, Sigma), NBQX (20 µM, N183, Sigma),
148
strychnine (50 µM, S8753, Sigma) and tetrodotoxin (1 µM, TTX, T-500, Alomone labs)
149
were dissolved in E3 medium. Picrotoxin (100 µM, P1675, Sigma) was dissolved in
150
DMSO. Larvae were embedded in agarose and drugs added to the bath solution and
151
injected in the brain ventricle (2-4 nl) using a picospritzer (PV820, World Precision
152
Instruments). Fish were tested before and 10 (TTX) or 30 (all others) minutes after drug
153
delivery. For behavior testing y264 ; UAS:SCN5 larvae, RFP expression was visually
154
confirmed at 4 dpf after anaesthetization with 0.03% tricaine (MS-222, Sigma). Larvae
155
were allowed to recover 40 h in E3 before testing.
156 157
Ablations. y264 ; UAS:epNTR-RFP, y269 ; UAS:epNTR-RFP and y269 ; UAS:nfsB-
158
mCherry larvae and RFP negative siblings (controls) were treated with 10 mM
159
metronidazole in E3 from 3-5 dpf. To detect cell death during genetic ablation, larvae
160
were immersed for 1 h in 8 μm PhiPhiLux G1D2 (OncoImmunin) and washed three times
161
before live imaging as previously described (Yokogawa et al. 2012). Laser ablations were
6
162
performed in Gt(T2KSAG)j1229a (J1229) larvae which express GFP in the Mauthner cell
163
(Burgess et al. 2009). Mauthner cells, or RoV3 neurons for controls, were ablated using a
164
Leica TCS-SP5 II 2-photon confocal microscope with a 20x 0.95 NA objective and an
165
800 nm laser. Immunolabeling against GFP confirmed successful ablation of 70% of
166
targeted cells. Only data from confirmed ablations was analyzed.
167 168
Statistical analysis. Analyses were performed using Gnumeric
169
(http://projects.gnome.org/gnumeric/). Data in figures and text are mean and s.e.m.
170 171
Results
172 173
EFPs induce well-coordinated swimming movements in larval zebrafish which strongly
174
resemble C-start escape responses triggered by acoustic stimuli (Figure 1A,B,
175
supplemental movie 1). Larvae show two types of responses to acoustic stimuli
176
distinguished by latency and kinematics (Burgess and Granato 2007). In contrast the vast
177
majority of EFP responses were initiated within 10 ms of the stimulus (Figure 1C). Since
178
both AC and DC EFPs have been used in behavioral assays in fish we tested the
179
behavioral responsiveness to both stimulus types. Responsiveness to EFPs was greater for
180
square (DC) field pulses compared to sinusoidal (AC) pulses of the same duration and
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amplitude (Figure 1D). Using long duration field pulses (10 ms) we observed that most
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EFP responses were evoked by the onset of the positive field potential, not the pulse
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offset (Figure 1E), and increasing the duration of the square pulse did not increase
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responsiveness (Figure 1F). Surprisingly larvae showed a faster reaction time to EFPs
7
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than to acoustic stimuli (Figure 1C inset, electric, 2.7±0.06 ms; acoustic, 5.1±0.07 ms,
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p
