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Teaching In The Laboratory
The zebrafish brain in research and teaching: a simple in vivo and in vitro
model for the study of spontaneous neural activity
R. Vargas, I. þ. Jóhannesdóttir, B. Sigurgeirsson, H. þorsteinsson, and K. Æ. Karlsson
Department of Biomedical Engineering, Reykjavik University, Reykjavik, Iceland
Received 14 September 2010; Accepted 19 January 2011
Vargas R, Jóhannesdóttir Iþ, Sigurgeirsson B, þorsteinsson
H, Karlsson KÆ. The zebrafish brain in research and teaching: a
simple in vivo and in vitro model for the study of sponta-
neous neural activity. Adv Physiol Educ 35: 188 –196, 2011;
doi:10.1152/advan.00099.2010.—Recently, the zebrafish (Danio
rerio) has been established as a key animal model in neuroscience.
Behavioral, genetic, and immunohistochemical techniques have been
used to describe the connectivity of diverse neural circuits. However,
few studies have used zebrafish to understand the function of cerebral
structures or to study neural circuits. Information about the techniques
used to obtain a workable preparation is not readily available. Here,
we describe a complete protocol for obtaining in vitro and in vivo
zebrafish brain preparations. In addition, we performed extracellular
recordings in the whole brain, brain slices, and immobilized nonanes-
thetized larval zebrafish to evaluate the viability of the tissue. Each
type of preparation can be used to detect spontaneous activity, to
determine patterns of activity in specific brain areas with unknown
functions, or to assess the functional roles of different neuronal groups
during brain development in zebrafish. The technique described offers
a guide that will provide innovative and broad opportunities to
beginner students and researchers who are interested in the functional
analysis of neuronal activity, plasticity, and neural development in the
zebrafish brain.
extracellular recordings; development; neural circuits
CURRENTLY, zebrafish (Danio rerio) are considered a fundamen-
tal model in genetics (19, 31), toxicology (14, 16, 26), phar-
macology (1), and physiopathology (5, 13, 27). Zebrafish are
being increasingly used in key areas of neuroscience, such as
neuropharmacology (1), neural development and regeneration
(3), behavioral neuroscience (23), and the study of neural
disease (2). The use of standard neuroanatomic and immuno-
histochemical techniques during the last decade has resulted in
detailed characterization of the organization of the zebrafish
nervous system and the distribution of different neurotransmit-
ter systems (17, 21, 24, 28, 30). Recently, researchers have
turned to functional studies, many of which have focused on
behavioral processes (4, 8, 9) and the electrophysiological
characteristics of the spinal motor system (11, 15, 29). A small
number of studies have focused on the electrical activity of
specific areas of the adult zebrafish brain, such as the olfactory
lobe (30, 33), retina (6, 22, 34), and telencephalon (18, 25).
Lately, techniques combining genetic, behavioral, and electro-
physiological approaches in zebrafish larvae have been intro-
duced. These have established the link between synaptic pro-
teins and visual acuity in zebrafish larvae (32). In addition, the
expression of photoswitchable channels and pumps has per-
mitted the remote manipulation of neurons, neural circuits, and
behavior in zebrafish larvae (8).
Therefore, zebrafish have become a widely used model, and
they have been used in the classroom to teach important
biological concepts, particularly in genetics and development
(12). The advantages of using zebrafish, which are an excellent
model for research and education, include low cost, low main-
tenance, rapid development, ease of genetic manipulation,
genomic similarities with humans, ease of external fertiliza-
tion, and the possibility to produce eggs and embryos contin-
uously with only a few fish. Other important features include
fast development, a clear pattern of development, transparency
at early ages, and the relatively large size of the embryos and
larvae, which facilitate manipulation. In addition, it is possible
to buy wild-type, mutant, and transgenic strains of zebrafish at
a low cost (35).
Currently, technical details concerning the techniques used
to obtain a workable preparation are not gathered or readily
available. In this article, we describe, in detail, in vitro and in
vivo protocols for obtaining brain preparations of larval and
adult zebrafish to study neural activity during development.
These preparations can be used for electrophysiological studies
of spontaneous or evoked activity in the zebrafish nervous
system, to describe the patterns of activity in specific brain
regions, and to track neural circuits during development. The
use of our technique, together with other approaches, such as
pharmacological tests and genetic manipulations, offers new
possibilities for the functional analysis of neuronal communi-
cation, plasticity, and neurodevelopment. Consequently, there
are an increased number of available resources for researchers
interested in zebrafish and the possibility to teach neurophys-
iology via a simple model.
METHODS
Larval and young adult zebrafish (D. rerio, strain AB-3, Zirc;
Fig. 1B) were housed in the zebrafish facility at Reykjavík University
in a zebrafish aquatic housing system (Aquatic Habitats, Apopka, FL).
The housing system is a self-contained bench-top unit with a capacity
of 740 zebrafish (1,35 l volume), 3 gallons/min recirculation system,
thermal control (28.5°C), ambient light control (fish were maintained
on a 14:10-h light-dark cycle), filters, and a ultraviolet sterilizer.
Zebrafish Brain Assay In Vitro
Whole brain preparation. Larval or young adult zebrafish (Fig. 1B)
were anesthetized in a solution of distilled water and 0.02% MS-222
(tricaine methanesulfonate, Sigma-Aldrich, St. Louis, MO). Animals
were fixed with insect pins in a Sylgard-covered petri dish containing
artificial cerebrospinal fluid (aCSF) that consisted of the following (in
mM): 131 NaCl, 2 KCl, 1.25 KH
2
PO
4
, 2 MgSO
4
, 10 glucose, 2.5
CaCl
2
, and 20 NaHCO
3
. The aCSF was maintained at room temper-
ature and a pH of 7.4. First, the skin and skull bones of the zebrafish
head were removed. After the brain was exposed, the cranial nerves
Address for reprint requests and other correspondence: R. Vargas, Reykja-
vik Univ., Menntvegur 1, Reykjavik 101, Iceland (e-mail: rafael@ru.is).
Adv Physiol Educ 35: 188 –196, 2011;
doi:10.1152/advan.00099.2010.
188 1043-4046/11 Copyright © 2011 The American Physiological Society
were cut, and the brain was extracted using microdissecting tweezers
and the tips of insect pins (Fig. 1, Cand D). Particular care was taken
not to damage the olfactory bulbs and telencephalon because they can
detach easily from the remainder of the brain. To avoid this, we began
the dissection by cutting at the level of the junction between the spinal
cord and brain stem. The brain stem was gently lifted with an insect
pin, and the ventral roots of the cranial nerves were cut. The optic
nerve was cut with small scissors before the whole brain was lifted for
the dissection of the olfactory lobe (see APPENDIX A). A large amount
of practice was necessary to gain expertise and to reduce tissue
damage. For the electrophysiological recordings, the whole brain was
fixed to the bottom of the recording chamber with Vetbond tissue
adhesive (3M, St. Paul, MN). The preparation was maintained in
aCSF that was bubbled with 95% O
2
and 5% CO
2
at room tempera-
ture. Extracellular recordings were done to evaluate the viability of
the tissue. Recordings were obtained from the brains for 6 h after
extraction.
Brain slice preparation. Young adult zebrafish were anesthetized in
a solution of cold distilled water and 0.02% MS-222 (Sigma-Aldrich)
and killed by decapitation. The head was immersed in 4 – 8°C aCSF
(bubbled continuously with 5% CO
2
in O
2
at pH 7.4). The skull was
removed to expose the brain, the cranial nerves were cut, and the brain
was carefully removed. After the brain was removed, it was immedi-
ately immersed and washed in 4 – 8°C aCSF for 2–3 min. To generate
slices, the brain was suspended in a block of 2% agarose gel (50%
distilled water and 50% aCSF, agarose type VII-A, Sigma-Aldrich)
and fixed to the holder with Vetbond tissue adhesive (3M). This
method was used because of the small size of the adult brain (3 mm
long, 2 mm thick, and 2.5 mm wide; Fig. 1D). It was not possible to
fix the brain with glue, which is common in large brain preparations.
A vibratome (HM650 V, Thermo Fisher Scientific, Waltham, MA)
that was programmed to a high vibration frequency and a slow blade
advance speed produced the best slices with long-term durability.
Recordings were obtained from the brain slices for 6 h. The slice
thickness ranged from 300 to 400 m, the slice orientation was mostly
transverse, and three slices were obtained from each brain. In some
instances, parasagittal and coronal slices were obtained (Fig. 2, Band C). The
orientation of slices was selected according to the area of interest. For
recovery, the slices were transferred to a holding reservoir filled with
aCSF that was bubbled with 95% O
2
and 5% CO
2
at room tempera-
ture for 1 h before their transfer to the recording chamber (see
APPENDIX C).
Staining
Adult zebrafish were first anesthetized and the brains were then
removed according to the procedure described above. Brains were
immersion fixed in 2% paraformaldehyde in 0.1 M PBS at 4°C for 24
h. After fixation, brains were washed and stored in 0.1 M PBS (pH
7.4). Coronal (Fig. 2, Aand C), transversal, and parasagittal brain
slices (50 –100 m) were generated using a vibratome (Vibratome
Microm HM650 V, Thermo Fisher Scientific). Subsequently, slices
were fixed to microscope slides and immersed in 1% methylene blue
in 0.1 M PBS (pH 7.4) for 3 min (see APPENDIX C). Brain sections were
washed three times for 1 min each and coverslipped. Slices were
visualized using an upright microscope (FN1 eclipse, Nikon Instru-
ments), and micrographs were obtained with a DS-L2 digital camera
(Nikon Instruments; Fig. 2C).
DiI, a soluble fluorescent dye that marks cell membranes retro-
gradely and anterogradely, was used to evaluate the possibility of
tracing neuronal circuits. DiI crystals were placed over specific
zones of fixed whole brains, and brains were maintained for 7–10
days at 4°C in 0.1 M PBS. Sections were obtained according to the
procedure described above. The localization and distribution of DiI
were examined using an epifluorescence microscope (FN1 eclipse,
Nikon Instruments) with a specific filter set (G-2B, excitation filter:
510/560 nm, barrier filter: 610 nm, Nikon Instruments; Fig. 2D).
Recording Technique
Recording electrodes were generated from borosilicate glass
(WPI, New Haven, CT) using a vertical puller (PC-10, Narishige
Group, Tokyo, Japan). A single-stage pull was used to make
pipettes with a long tapered end and with tip resistances of 1–2 M⍀
(5-to 10-m-wide tips). An upright microscope (Nikon Eclipse
FN1, Nikon, Tokyo, Japan) and micromanipulator assembly
(MHW-3, Narishige Group) were used to obtain the recordings.
Microelectrodes filled with aCSF were lowered into the tissue
while a positive pressure was applied through the pipette tip using
a 10-ml syringe. The microelectrode was carefully inserted into the
tissue under visual guidance (Figs. 1C,3C,4A, and 5B). A seal test
(brief voltage steps of 1 mV, 10 ms, 100 Hz) permitted observation
of the electrode during its advancement. Upon noting a decrease in
current (reflecting an increase in resistance as a cell was ap-
proached), the pressure at the pipette tip was reversed via the
application of mild suction through the syringe. Seal resistances in
Fig. 1. Zebrafish model. A: photograph showing a zebrafish
embryo at 3 days postfertilization. The embryo is in its chorion.
Calibration bar ⫽200 m. B: a 5-days postfertilization larva
(top) and 5-mo-old adult zebrafish (bottom). Calibration bar ⫽
5mm.C: whole brain of a 5-days postfertilization larva with a
recording electrode over the telencephalon (Tel). TO, tectum
opticum; BS, brain stem. Calibration bar ⫽100 m. D: brain
of a 5-mo-old adult zebrafish (ventral side). Calibration bar ⫽
300 m. OL, olfactory lobe; HP, hypothalamus.
Teaching In The Laboratory
189SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
the range of 10 –20 M⍀were obtained, and recordings were
collected for up to several hours. The recordings were amplified
and filtered (3 Hz–10 kHz) through a Multiclamp 700B amplifier
(MDS Analytical Technologies, Sunnyvale, CA) and digitized with
a data-acquisition interface Power 1401 analog-to-digital converter
(CED, Cambridge, UK). Extracellular recordings were obtained
from the following different areas of the whole brain: tectum
opticum, telencephalon, and brain stem. We emphasized the tel-
encephalon because we were particularly interested in this struc-
ture (Figs. 1Cand 3C). The acquisition and analysis of signals
were performed using Signal 4 software (CED). Additionally, we
tested the effect of a chemical stimulus on telencephalic electrical
activity, i.e., caffeine (1 mM), which is a general-purpose excit-
atory agent (10).
Zebrafish Brain Assay In Vivo
Zebrafish larvae were anesthetized in a solution of distilled water
and 0.02% MS-222 (Sigma-Aldrich). Larvae were suspended in 2%
agarose gel (type VII-A, Sigma-Aldrich), and each embeded larva was
fixed to the bottom of a petri dish with Vetbond tissue adhesive (3M)
in circulating aCSF at room temperature. Next, the soft tissue was
removed to expose the following brain structures: the telencephalon,
tectum opticum, and cerebellum (Figs. 5, Aand B). Using this method,
larvae survived for ⬎1 h (see APPENDIX B).
Statistical Analysis
Spontaneous electrical activity was analyzed in recordings obtained
from the telencephalon of adult whole zebrafish brains under basal
Fig. 2. Zebrafish brain slices. A: fixed coronal section of a
5-days postfertilization larva showing the telencephalon in
situ.B: brain slice of a 5-mo-old adult telencephalon (para-
sagittal section). DT, dorsal telencephalon; VT, ventral
telencephalon; ADL, anterior dorsal lobe. C: fixed brain
slice of a 5-mo-old adult telencephalon (transverse section)
stained with methylene blue. The inset shows the cellular
density in the entopeduncular nucleus (EN). D: fixed brain
slice from a 5-mo-old adult showing the telencephalon
(coronal section). *Location of the DiI stain. The inset
shows labeled fibers in the telencephalon (epifluorescence
microscopy). Calibration bars ⫽100 m(A–D) and 50 m
(insets).
Fig. 3. Electrical activity in the adult zebrafish whole brain in
vitro (ventromedial telencephalon preparation). A: 4-mo-old
adult zebrafish brain in situ. Ce, cerebellum. B: whole brain in
vitro (ventral side). C: ventral side of the telencephalon with
a recording microelectrode over the ventral medial side.ON,
optic nerve; OB, oflactory bulb. Calibration bars ⫽300 m.
D–F: the traces show the recorded spontaneous electrical
activity in the telencephalon. The horizontal bar in the top
trace (D) indicates the segment of recording that was ex-
panded in the middle trace (E), and the horizontal bar in the
middle trace indicates the segment of recording that was
expanded in the bottom trace (F).
Teaching In The Laboratory
190 SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
conditions and after exposure to 1 mM caffeine (Fig. 6, A–D).
Averages of rate, amplitude, and duration of spike-like events were
evaluated before and after caffeine administration. The effect of
caffeine was analyzed using Student’s paired t-test.
RESULTS
Zebrafish Brain Assay In Vitro
Whole brain preparation. The in vitro preparation was
performed for both larvae and adults (Figs. 1, Cand D, and 3,
A–C). Dorsal and ventral areas were explored on one side of
the isolated whole brain in each experiment because the brain
was fixed with adhesive tissue to the chamber. Therefore, we
explored dorsal areas of the telencephalon and tectum opticum
and ventral areas of the telencephalon and brain stem (36) (Fig.
3C). The brain dissection was not complicated, and the viabil-
ity of the preparation was high (⬎90%). In adult brains, the
size and tissue opacity made it difficult to explore deeper zones
such as the thalamus and brain stem nuclei; therefore, a slice
preparation was required (Figs. 3, A–C and 4A). Spontaneous
basal activity was found in all areas of the brain as rhythmic
oscillatory waves or as discrete spike-like events. This activity
was absent in damaged brains.
Brain slice preparation. To generate the slices, the brain was
suspended in a block of agarose type VII-A and fixed to the
holder with cyanoacrylate glue. This method was used because
of the small size of the adult brain (3 mm long, 2 mm thick, and
2.5 mm wide; Figs. 1Dand 3, A–C). It was not possible to fix
the brain with glue, which is a commonly used procedure in
large brain preparations. Agarose was prepared using 50%
distilled water and 50% aCSF to avoid hyperosmolarity, which
Fig. 4. Electrical activity in the adult zebrafish in transversal
brain slices of the tectum opticum. A: transverse slice obtained
from a 4-mo-old adult zebrafish with the recording microelec-
trode over the tectum opticum. T, thalamus. Calibration bar ⫽
100 m. B–D: the traces show the recorded spontaneous
electrical activity. The horizontal bar in the top trace (B)
indicates the segment of recording that was expanded in the
middle trace (C), and the horizontal bar in the middle trace
marks the segment of recording that was expanded in the
bottom trace (D).
Fig. 5. Electrical activity in zebrafish larvae in vivo.Aand B:
photographs showing a larva (5 days postfertilization; A) and a
larval whole brain (5 days postfertilization) in vivo with the
recording microelectrode over the tectum opticum. Calibration
bar ⫽100 m. C–E: extracellular recordings obtained in the
5-days postfertilization larval whole brain in vivo. The traces
show the recorded spontaneous electrical activity. The hori-
zontal bar in the top trace (C) indicates the segment that was
expanded in the middle trace (D), and the horizontal bar in the
middle trace marks the segment that was expanded in the
bottom trace (E).
Teaching In The Laboratory
191SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
causes the tissue to become nonviable. A high vibration fre-
quency and a slow blade advance speed produced viable slices
with thicknesses that ranged from 300 to 400 m. Brain slices
were conserved for 6 h. Three slices were obtained in the
transverse orientation (n⫽3; Fig. 4), and parasagittal (n⫽2)
and coronal (n⫽2) slices were also obtained. The slice
orientation was selected according to the area of interest.
Staining. To identify the structural organization of the ze-
brafish brain, we used a simple histological protocol with 1%
methylene blue, which is a dye that marks cellular bodies of
neurons in fixed brain slices (Fig. 2C). In some cases, larvae
were fixed and stained according to the procedure used for
adult brains (Fig. 2A). To evaluate the possibility of tracing
neuronal circuits and neuronal projections, we used DiI, a
soluble fluorescent dye that marks cell membranes retrogradely
and anterogradely (Fig. 2D). The application of this marker
was simple, and the circuits were visible after 1 wk of brain
incubation with the dye; no additional treatment was necessary
to obtain slices from brains marked with DiI.
Zebrafish Brain Assay In Vivo
The in vivo brain preparation was performed only in larvae.
Larvae were submerged in agarose gel, in which they survived
for ⬎1 h. The recorded brain regions included the dorsal
telencephalon, tectum opticum, and cerebellum (Fig. 5, Aand
B). These areas were easily exposed by removal of the sur-
rounding soft tissue with little damage. To accomplish this
dissection, the tip of a micropipette or an insect pin was used
as a scalpel with excellent results (see APPENDIX B).
Recording Technique
In general, spontaneous electrical activity was detected in
all brain regions explored: the telencephalon (n⫽5, activity
in 80% of brains explored), tectum opticum (n⫽5, activity in
40% of brains explored), and brain stem (n⫽5, activity in
80% of brains explored). The olfactory lobe demonstrated
spontaneous activity that had a rhythmic, oscillatory pattern or
displayed spikes. In some cases, the oscillatory activity showed
an amplitude-modulated pattern. This type of activity has been
previously reported in a study (7) of the olfactory neuroepi-
thelium. Spontaneous electrical activity was more frequent and
easier to detect in larval brains than in young adult brains.
Spontaneous spikes were observed in the telencephalon, tec-
tum opticum, and brain stem, and rhythmic activity was ob-
served in the olfactory lobe, tectum opticum, and cerebellum.
Spontaneous activity was not detected in brains with mild
damage.
In the adult slice preparation, spontaneous electrical activity,
which again appeared as a rhythmic, oscillatory pattern or as
spikes (Fig. 4), was not consistently present.
In our laboratory, we are interested in exploring the spon-
taneous electrical activity produced by the telencephalon.
Therefore, in the present study, we emphasized the recording
of spontaneous electrical activity in the telencephalon of young
adult zebrafish brains. Spontaneous electrical activity became
visible as spikes (Fig. 3D). To evaluate tissue excitability in the
telencephalon, we tested the effect of a chemical stimulus on
telencephalic activity using caffeine (1 mM), which is a gen-
eral-purpose excitatory drug. Caffeine increased the frequency
and amplitude of spikes (Fig. 6). After the application of
caffeine, the spike frequency increased from 0.029 ⫾0.01 to
0.154 ⫾0.05 Hz [(n⫽7, time (t)⫽⫺2.6, P⫽0.041)], the
amplitude increased from 0.036 ⫾0.01 to 0.107 ⫾0.031 nA
(n⫽7, t⫽⫺3.1, P⫽0.021), and the duration increased from
88.7 ⫾23.3 to 165.4 ⫾19.4 ms (n⫽7, t⫽⫺4.4, P⫽0.005).
DISCUSSION
In the present article, we describe the preparation of ze-
brafish larvae in vivo and of the brain of zebrafish of different
ages in vitro (from the larval stage to the young adult). These
preparations are viable for many hours (a minimum of1hin
the present study). The zebrafish brain is transparent during
early development but not in adults (Figs. 1Dand 3, A–C).
Therefore, it is practical to use a slice preparation of the adult
brain. We demonstrate that it is possible to obtain slices from
the adult zebrafish brain that are viable for hours (Fig. 4, A–D).
After standardizing the protocols for the in vitro (APPENDIX
A–C) and in vivo (APPENDIX B) models, we explored the electri-
cal activity that is produced by the zebrafish brain. We ob-
served spontaneous electrical activity in the youngest larvae at
5 days postfertilization (Fig. 5, A–E). Spontaneous basal ac-
tivity was found in all areas of the brain as rhythmic oscillatory
waves or as discrete spike-like events; this latter activity could
represent excitatory or inhibitory postsynaptic currents. Typi-
cally, more prominent spontaneous activity is observed in
younger brains compared with adult brains. The differences in
neural activity between young and adult brains may be ex-
plained by differences in maturation; for example, the high
proportion of glial and endothelial cells in adult brains results
in difficulties of obtaining clean recordings due to a decrease in
the signal-to-noise ratio. Furthermore, the maturation of dif-
Fig. 6. Effect of caffeine on spontaneous electrical activity in
the telencephalon. Electrical activity in the anterior dorsal lobe
of the telencephalon in adult brains was recorded before (A)
and after (B) caffeine application (1 mM) in the artificial
cerebrospinal fluid (aCSF) bath solution. A: regular spontane-
ous activity, which was detected as spikes, was observed at
rest. B: caffeine (1 mM) increased the frequency, amplitude,
and duration of the recorded spikes. A–D: the horizontal bars
in the top traces (Aand B) indicate the segments that were
expanded in the middle traces (Cand D). *Segment that were
expanded in the bottom traces (Eand F).
Teaching In The Laboratory
192 SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
ferent neurotransmitter systems could give rise to differences
in the activation and deactivation of specific neural circuits.
These phenomena should be studied in detail using a variety of
techniques. Brains that exhibited severe damage or were ob-
tained from dead animals did not demonstrate electrical activ-
ity. A great amount of practice was necessary to gain expertise
in the tissue dissection and to reduce tissue damage.
Traditionally, the classical models used to teach electrophys-
iology use frogs, chickens, and rats. The most common de-
monstrative experiments that are performed in colleges and
schools use the sciatic nerve and gastrocnemius muscle to
explain the biophysical properties of muscles and neurons and
synaptic transmission at the neuromuscular plate. These mod-
els are used to teach concepts such as membrane potential,
resting potential, action potential, latency, threshold potential,
conduction velocity, and synaptic transmission at the neuro-
muscular plate and its associated properties. In only a few
cases has the brain been used to teach central nervous system
phenomena. In general, brain activity has been explored by
behavioral experiments (20). Electrophysiology instruction us-
ing the brain has been difficult because rats, mice, and chickens
are expensive, difficult to manipulate, and, in many cases,
require specific temperature conditions. The large size of their
brains also necessitates the use of brain slice preparations for
the experiments.
Using a pilot training program, we have trained graduate and
postgraduate students attending a biomedical engineering pro-
gram in the different steps needed to acquire expertise in a
complete method for extracting zebrafish brain (details in
APPENDIX A–C) and performing basic electrophysiological re-
cordings (see APPENDIX D). After practical training, students
without a previous biological background in neuroscience
gained the skills necessary to perform a complete experiment.
The procedures outlined in this article can be replicated at very
low cost, with custom-built equipment. Researchers, universi-
ties, and research institutions around the world, especially in
developing countries, can establish these protocols quickly and
easily to the benefit of present and future generations of
students and researchers. Although our goal with the use of the
brain extraction method has been to obtain electrophysiological
recordings, we have used the brain extraction protocol in
histological, genetic, and neuroplasticity studies. Clearly, the
zebrafish is an ideal model for teaching and research, because
it permits students to develop skills in areas such as small-
animal surgery, neuroanatomy, and neurophysiology. Addi-
tionally, in research, the brain extraction technique is funda-
mental for neurophysiology, immunohistochemistry, neuro-
genesis, neurogenetics, and neurochemistry studies.
Therefore, we propose that the zebrafish brain be used as an
inexpensive and accessible model to study the electrophysio-
logical phenomena that occur in the central nervous system and
the activity of neural circuits.
APPENDIX A: ADULT ZEBRAFISH WHOLE BRAIN IN VITRO
Materials
The following materials are needed:
•Adult zebrafish
•Beakers (100 and 300 ml)
•Insulin syringes
•Needles (21 gauge, 1 in.)
•Microtweezers
•Microscissors (3¼ in.)
•Glass microcapillaries
•Pasteur pipettes (1–2 ml)
•Plastic petri dishes
•Plastic box and sponge
•Spoon
•Dissecting stereomicroscope
aCSF
aCSF is composed of the following:
•Distilled water (100 ml)
•NaCl (7,656 g)
•KCl (0.0149 g)
•KH
2
PO
4
(0.017 g)
•MgSO
4
·7 H
2
O (0.0493 g)
•Glucose (0.1802 g)
•CaCl
2
(0.0277 g)
•NaHCO
3
(0.1608 g)
aCSF is bubbled with a gas mixture of 95% O
2
-5% CO
2
(pH 7.4).
Anesthetic
The anesthetic is MS-222 (3-aminobenzoic acid ethyl ester meth-
anesulfonate salt). The stock solution is 2%. It is composed of the
following:
•Distilled water (100 ml)
•MS222 powder (2 g)
Protocol
The protocol for adult zebrafish brain extraction is shown in Fig. 7.
APPENDIX B: LARVAL ZEBRAFISH BRAIN IN VITRO AND
IN VIVO
Materials
The following materials are needed:
•Larval zebrafish
•Beakers [20 ml (agarose), 100 ml (anesthetic), and 300 ml (aCSF)]
•Insulin syringes
•Insect pins
•Glass microcapillaries
•Pasteur pipettes (1–2 ml)
•Plastic petri dishes
•Cyanoacrilate glue (Vetbond)
•Dissecting stereomicroscope
Solutions
The following solutions are used:
•aCSF (100 ml)
•MS-222 (0.02%, 100 ml)
Agarose
Agarose is composed of the following:
•Low-melting agarose type VII-A powder (200 mg)
•aCSF (5 ml)
•Distilled water (5 ml)
Protocol
The protocol for larval zebrafish brain extraction is shown in Fig. 8.
Teaching In The Laboratory
193SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
APPENDIX C: BRAIN SLICE PREPARATION
Materials
The following materials are needed:
•Adult zebrafish
•Beakers [20 ml (agarose), 100 ml (anesthetic), and 300 ml (aCSF)]
•Insulin syringes
•Needles (21 gauge, 1 in.)
•Microtweezers
•Microscissors (3¼ in.)
•Glass microcapillaries
•Pasteur pipettes (1–2 ml)
•Plastic petri dishes
•Plastic box and sponge
•Spoon
•Cyanoacrilate glue (Vetbond)
•Dissecting stereomicroscope
•Vibratome
•Petri dish with nylon net
•Gas mixture of 95% O
2
and 5% CO
2
Fig. 7. Protocol for adult zebrafish brain extraction. A: the instruments used for the dissection are inexpensive and include syringe needles, glass microcapillaries,
insect pins, petri dishes, pipettes, scalpels, tweezers, and microscissors. B: initially, adult zebrafish are anesthetized in a solution of distilled water and 0.02%
MS-222. A Sylgard petri dish or box with sponge are used to hold the fish during the brain extraction. A sponge is commonly used to fix the fish during
intraperitoneal drug microinjections. C: spoons are used to manipulate the fish and avoid injury. D: the fish is fixed with insect pins or syringe needles in a
Sylgard-covered petri dish containing aCSF. E: the skin and skull bones of the zebrafish head are removed from one side to avoid telencephalon damage. F: the
other side of the skull is removed to expose the complete brain; the telencephalon, tectum opticum, and cerebellum are now clearly visible. G: the spinal cord
is cut with a syringe needle at the junction between the spinal cord and brain stem. The brain is then lifted with a capillary glass, and the cranial nerves are exposed
and cut. H: the optic nerve (*) is cut with small scissors before the whole brain is lifted for dissection of the olfactory lobe. Particular care is taken not to damage
the olfactory bulbs and telencephalon because they can detach easily from the remainder of the brain. I: finally, the brain is extracted using microdissecting
tweezers or the tips of insect pins. The brain is moved from one place to another using a Pasteur pipette. For electrophysiological recordings, the whole brain
is fixed to the bottom of the recording chamber with Vetbond tissue adhesive. The preparation is maintained in aCSF that is bubbled with 95% O
2
and 5% CO
2
at room temperature.
Fig. 8. Protocol for larval zebrafish brain extraction. A: the
instruments are inexpensive and include the following: nee-
dles, glass microcapillaries, insect pins, and petri dishes. We
used MS-222 as the anesthetic. B: initially, zebrafish larvae are
anesthetized in a solution of distilled water and 0.02% MS-
222. C: the larva is suspended in a 2% agarose gel, and each
agarose-embedded larva is fixed to the bottom of a petri dish
with Vetbond tissue adhesive in circulating aCSF at room
temperature. D: agarose is removed to expose the head of the
larva. E: glass capillaries (*) or insect pins are used as scalpels.
The soft tissue is removed. Fand G: the following brain
structures are exposed: the telencephalon, tectum opticum,
cerebellum, and brain stem. When we work with in vivo
preparations we stop the dissection at this point; for brain
extraction, we continue with the next step. H: we cut the spinal
cord with a capillary tip at the junction between the spinal cord
and brain stem. The brain stem is gently lifted, and the roots of
the cranial nerves are cut with glass capillaries. I: finally, after
the optic nerve and olfactory nerve are cut, we extract the
complete brain.
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194 SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
Solutions
The following solutions are used:
•aCSF (100 ml)
•MS-222 (0.02%, 100 ml)
Agarose
Agraose is composed of the following:
•Low-melting agarose type VII-A (200 mg)
•aCSF (10 ml)
Protocol
The protocol for adult zebrafish brain slices is shown in Fig. 9. The
process for adult brain extraction is the same as described in APPENDIX
A; here, the difference is that we used cold solutions during the
process. Adult zebrafish are anesthetized in a solution of cold (4 – 8°C)
distilled water and 0.02% MS-222 (Sigma-Aldrich) and killed by
decapitation. The head is immersed in 4 – 8°C aCSF, which is bubbled
continuously with 5% CO
2
in O
2
. The skull is removed to expose the
brain, the cranial nerves are cut, and the brain is carefully removed
(see APPENDIX A). After the brain is removed, it is immediately
immersed and washed in 4 – 8°C aCSF for 2–3 min.
Next, to generate the slices, the brain is suspended in a block of
agarose (type VII-A) and fixed to the holder with cyanoacrylate glue.
Slices are obtained with a vibratome (HM650 V) programmed to a
high vibration frequency and a slow blade advance speed to produce
the best slices (300 – 400 m) with long-term durability. For recovery,
the slices must be transferred to a holding reservoir filled with aCSF
bubbled with 95% O
2
-5% CO
2
at room temperature for 1 h before
their transfer to the recording chamber.
For histology, adult zebrafish are first anesthetized, and the brain is
then removed according to the procedure described above. The brain
is immersion fixed in 2% paraformaldehyde in 0.1 M PBS at 4°C for
24 h. After fixation, the brain is washed and stored in 0.1 M PBS (pH
7.4). The brain is suspended in a block of 2% agarose gel (0.1 M PBS,
agarose type VII-A, Sigma-Aldrich). Slices are obtained using the
protocol described above.
APPENDIX D: BASIC GUIDELINES FOR EFFECTIVE
PRACTICE FOR THE INSTRUCTOR
The goal of this outline is to facilitate the implementation of a
workshop for undergraduate and graduate students and young scien-
tists who are interested in zebrafish brain research. The course is
organized in five modules that can be scheduled individually or
consecutively. In addition, the course can be carried out as an
intensive short-term or long-term course and for training that includes
a major emphasis on experimental strategies and the manipulation of
instruments and equipment.
The training starts with a practice class explaining the basic
concepts of the laboratory instruments and their manipulation, the
preparation of solutions, optic microscopy, and the manipulation of
stereomicroscopy and upright microscopy. This instruction is fol-
lowed by the preparation of anesthetics and surgery for brain extrac-
tion. The next step comprises the preparation of microelectrodes for
recording, practice with the micromanipulators, and familiarization
with the amplifier, signal conditioner, and software used for the
acquisition and analysis of signals. Finally, a complete electrophysi-
ological recording is performed. The practice is complemented by
instructions concerning the slice preparation.
Modules
Module 1: laboratory instrument manipulation and preparation of
solutions. In this part of the course, the following instruments are
used: micropipettes, scale, stirrer, vortex, glass, and salts. Instructions
regarding the use of these instruments are followed by the manipula-
tion and preparation of the aCSF. Students will also learn to make
drug stock solutions and dilutions.
Module 2: training in the use of the stereomicroscope and upright
microscope and brain extraction. The aim of this part of the course is
to acquire skill with respect to the use of microscopes. To achieve this
aim, students will practice using the stereomicroscope and upright
microscope. This practice is followed by the preparation of the
anesthetic and extraction of a zebrafish brain from a young adult fish
using the following surgical instruments: scalpels, tweezers, needles,
insect pins, and petri dishes.
Module 3: microelectrode preparation and set up of the in vitro
brain preparation. This part of the course is dedicated to the prepa-
ration of glass microelectrodes for recordings using the puller and
polisher. In addition, the students will learn to set up the in vitro brain
preparation using the bath chamber and holder, to fill the microelec-
trodes with aCSF for extracellular recordings, and to fix the brains to
the preamplifier head stage.
Module 4: electrophysiological recordings in the brain. This part
of the course is dedicated to performing the extracellular recordings.
The students will learn to manipulate the micromanipulators, upright
Fig. 9. Protocol for adult zebrafish brain slices. A: to prepare
slices, we use a vibratome with a stereomicroscope attached to
improve visualization of the tissue. B: to generate slices, the
brain is suspended in a block of 2% agarose gel. C: the agarose
block with the brain is fixed to the holder (H) with Vetbond
tissue adhesive. This method is used because of the small size
of the adult brain (3 mm long, 2 mm thick, and 2.5 mm wide).
It is not possible to fix the brain with glue, which is a common
procedure used in large brain preparations. D: the holder is
then attached to the vibratome tray, which contains aCFS at
4°C. Eand F: the vibratome is programmed to a high vibration
frequency and a slow blade (B) advance speed to produce the
best slices with long-term durability. G: the slice thickness
ranges from 300 to 400 m. H: the slice orientation is
according to experimental requirements. Here, we show a
transverse slice. In this case, 3 slices (300 –400 m) were
obtained from each brain. I: for recovery, the slices must be
incubated in aCSF aerated with 95% O
2
and 5% CO
2
at room
temperature for 1 h before their transfer to the recording
chamber. To avoid tissue damage by bubble movements, slices
are placed in a petri dish with a nylon net.
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195SPONTANEOUS AND EVOKED ELECTRICAL ACTIVITY IN THE ZEBRAFISH BRAIN
Advances in Physiology Education •VOL 35 •JUNE 2011
microscope, amplifier and signal conditioner, software for acquisition,
and signal analyzer.
Module 5: slices and staining. The goal of this part of the course is
to acquire skills with the vibratome and to prepare brain slices for
recordings and staining. For the electrophysiological recordings, the
students will extract the brain and generate slices while the brain is
supported in agarose blocks. For the staining procedure, the brains are
extracted and fixed in 2% paraformaldehyde in 0.1 M PBS. Slices can
be used immediately or saved for the fluorescent dye experiments. In
DiI experiments, the brain is maintained in 2% paraformaldehyde in
0.1 M PBS solution for a minimum of 7 days, and brain slices are
generated at later time points. The epifluorescence microscope is used
with specific filters for the DiI marker to observe the traced circuits.
Advanced students and researchers can find more detailed infor-
mation about sophisticated electrophysiological techniques in the
REFERENCES, which include intracellular recordings, Ca
2⫹
imaging,
channelrodopsin, and photoactivation (4, 6, 8, 11, 32, 34). Also,
additional information can be found on the use of slices for immuno-
histochemistry, tracing neural circuits with fluorescent dyes and green
fluorescent protein expression (17, 21, 24, 28), and genetic studies
(17, 31, 32).
GRANTS
This work was supported by Icelandic Centre for Research Grant
080441022-091012 (to K. Æ. Karlsson).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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