Larval zebrafish are an appropriate animal and laboratory model for exploring the neural mechanisms underlying cognitive abilities, especially concerning their applicability to human cognition. To replicate the natural habitats of such organisms at the laboratory level, microfluidic platforms are employed as a valuable tool in mimicking the intricate spatiotemporal stimuli together with high-throughput screening. This work investigated the memory capabilities of zebrafish larvae across different developmental stages (5–9 days post-fertilization) by employing sound stimuli within the microfluidic environment. Notably, the sound signal with 1200 Hz frequency was observed to be significantly sensitive among all the considered developmental stages in stimulating the responses. In addition, the impact of the memory enhancer drug methylene blue (MB) was tested, revealing a significant enhancement in cognitive performance compared to controls. Specifically, learning (training) and memory (post-training) were observed to exhibit 2-fold and 20-fold increases, respectively, in MB-exposed larvae. In addition to sound stimuli and memory enhancer drugs, the impact of environmental complexity on cognitive abilities was examined by employing different designs of microchannels, such as series, parallel, and combined configurations. The presented experimental paradigm provides a robust framework for various zebrafish studies, including sensory processing mechanisms, learning capabilities, and potential therapeutic interventions.

1.
E. E.
Patton
,
L. I.
Zon
, and
D. M.
Langenau
,
Nat. Rev. Drug Discovery
20
(
8
),
611
628
(
2021
).
2.
K.
Howe
,
M. D.
Clark
,
C. F.
Torroja
,
J.
Torrance
,
C.
Berthelot
,
M.
Muffato
,
J. E.
Collins
,
S.
Humphray
,
K.
McLaren
,
L.
Matthews
,
S.
McLaren
,
I.
Sealy
,
M.
Caccamo
,
C.
Churcher
,
C.
Scott
,
J. C.
Barrett
,
R.
Koch
,
G.-J.
Rauch
,
S.
White
,
W.
Chow
,
B.
Kilian
,
L. T.
Quintais
,
J. A.
Guerra-Assunção
,
Y.
Zhou
,
Y.
Gu
,
J.
Yen
,
J.-H.
Vogel
,
T.
Eyre
,
S.
Redmond
,
R.
Banerjee
,
J.
Chi
,
B.
Fu
,
E.
Langley
,
S. F.
Maguire
,
G. K.
Laird
,
D.
Lloyd
,
E.
Kenyon
,
S.
Donaldson
,
H.
Sehra
,
J.
Almeida-King
,
J.
Loveland
,
S.
Trevanion
,
M.
Jones
,
M.
Quail
,
D.
Willey
,
A.
Hunt
,
J.
Burton
,
S.
Sims
,
K.
McLay
,
B.
Plumb
,
J.
Davis
,
C.
Clee
,
K.
Oliver
,
R.
Clark
,
C.
Riddle
,
D.
Elliott
,
G.
Threadgold
,
G.
Harden
,
D.
Ware
,
S.
Begum
,
B.
Mortimore
,
G.
Kerry
,
P.
Heath
,
B.
Phillimore
,
A.
Tracey
,
N.
Corby
,
M.
Dunn
,
C.
Johnson
,
J.
Wood
,
S.
Clark
,
S.
Pelan
,
G.
Griffiths
,
M.
Smith
,
R.
Glithero
,
P.
Howden
,
N.
Barker
,
C.
Lloyd
,
C.
Stevens
,
J.
Harley
,
K.
Holt
,
G.
Panagiotidis
,
J.
Lovell
,
H.
Beasley
,
C.
Henderson
,
D.
Gordon
,
K.
Auger
,
D.
Wright
,
J.
Collins
,
C.
Raisen
,
L.
Dyer
,
K.
Leung
,
L.
Robertson
,
K.
Ambridge
,
D.
Leongamornlert
,
S.
McGuire
,
R.
Gilderthorp
,
C.
Griffiths
,
D.
Manthravadi
,
S.
Nichol
,
G.
Barker
,
S.
Whitehead
,
M.
Kay
,
J.
Brown
,
C.
Murnane
,
E.
Gray
,
M.
Humphries
,
N.
Sycamore
,
D.
Barker
,
D.
Saunders
,
J.
Wallis
,
A.
Babbage
,
S.
Hammond
,
M.
Mashreghi-Mohammadi
,
L.
Barr
,
S.
Martin
,
P.
Wray
,
A.
Ellington
,
N.
Matthews
,
M.
Ellwood
,
R.
Woodmansey
,
G.
Clark
,
J. D.
Cooper
,
A.
Tromans
,
D.
Grafham
,
C.
Skuce
,
R.
Pandian
,
R.
Andrews
,
E.
Harrison
,
A.
Kimberley
,
J.
Garnett
,
N.
Fosker
,
R.
Hall
,
P.
Garner
,
D.
Kelly
,
C.
Bird
,
S.
Palmer
,
I.
Gehring
,
A.
Berger
,
C. M.
Dooley
,
Z.
Ersan-Ürün
,
C.
Eser
,
H.
Geiger
,
M.
Geisler
,
L.
Karotki
,
A.
Kirn
,
J.
Konantz
,
M.
Konantz
,
M.
Oberländer
,
S.
Rudolph-Geiger
,
M.
Teucke
,
C.
Lanz
,
G.
Raddatz
,
K.
Osoegawa
,
B.
Zhu
,
A.
Rapp
,
S.
Widaa
,
C.
Langford
,
F.
Yang
,
S. C.
Schuster
,
N. P.
Carter
,
J.
Harrow
,
Z.
Ning
,
J.
Herrero
,
S. M. J.
Searle
,
A.
Enright
,
R.
Geisler
,
R. H. A.
Plasterk
,
C.
Lee
,
M.
Westerfield
,
P. J.
de Jong
,
L. I.
Zon
,
J. H.
Postlethwait
,
C.
Nüsslein-Volhard
,
T. J.
Hubbard
,
H.
Roest Crollius
,
J.
Rogers
, and
D. L.
Stemple
,
Nature
496
(
7446
),
498
503
(
2013
).
3.
V.
Surendra
,
U. R.
Sharma
, and
D.
Goli
, “
Behavioral studies of different drugs using zebrafish as a model
,”
Int. J. Pharm.
2
,
75
82
(
2011
).
4.
J. R.
Fetcho
and
K. S.
Liu
,
Ann. N. Y. Acad. Sci.
860
,
333
345
(
1998
).
5.
F. M.
Richards
,
W. K.
Alderton
,
G. M.
Kimber
,
Z.
Liu
,
I.
Strang
,
W. S.
Redfern
,
J. P.
Valentin
,
M. J.
Winter
, and
T. H.
Hutchinson
,
J. Pharmacol. Toxicol. Methods
58
(
1
),
50
58
(
2008
).
6.
K. M.
Khan
,
A. D.
Collier
,
D. A.
Meshalkina
,
E. V.
Kysil
,
S. L.
Khatsko
,
T.
Kolesnikova
,
Y. Y.
Morzherin
,
J. E.
Warnick
,
A. V.
Kalueff
, and
D. J.
Echevarria
,
Br. J. Pharmacol.
174
(
13
),
1925
1944
(
2017
).
7.
Y.
Xi
,
S.
Noble
, and
M.
Ekker
,
Curr. Neurol. Neurosci. Rep.
11
(
3
),
274
282
(
2011
).
8.
D. S.
Wiley
,
S. E.
Redfield
, and
L. I.
Zon
,
Methods Cell Biol.
138
,
651
679
(
2017
).
9.
W. F.
An
and
N.
Tolliday
,
Mol. Biotechnol.
45
(
2
),
180
186
(
2010
).
10.
S.
Gimondi
,
H.
Ferreira
,
R. L.
Reis
, and
N. M.
Neves
,
ACS Nano
17
(
15
),
14205
14228
(
2023
).
11.
V.
Sahadevan
,
D.
Loganathan
,
Y.
Chuang
,
C.
Feng Lo
,
C.-Y.
Chen
, and
C.-Y.
Chen
,
Mater. Chem. Phys.
307
,
128068
(
2023
).
12.
S.
Aralekallu
,
R.
Boddula
, and
V.
Singh
,
Mater. Des.
225
,
111517
(
2023
).
13.
B.
Panigrahi
and
C.-Y.
Chen
,
Micromachines
10
(
12
),
880
(
2019
).
14.
M. D. O.
Barreiros
,
F. G.
Barbosa
,
D. D. O.
Dantas
,
D. D. M. L. D.
Santos
,
S.
Ribeiro
,
G. C. D. O.
Santos
, and
A. K.
Barros
,
Sci. Rep.
11
(
1
),
9330
(
2021
).
15.
C.-Y.
Chen
and
C.-M.
Cheng
,
Adv. Healthcare Mater.
3
(
6
),
940
945
(
2014
).
16.
F.
Yang
,
C.
Gao
,
P.
Wang
,
G.-J.
Zhang
, and
Z.
Chen
,
Lab Chip
16
(
7
),
1106
1125
(
2016
).
17.
M. D. A. M. M.
Ferraz
and
G. D. A.
Ferronato
,
Anim. Reprod.
20
(
2
),
e20230058
(
2023
).
18.
K.
Mani
and
C.-Y.
Chen
,
Microfluid. Nanofluid.
25
(
3
),
22
(
2021
).
19.
D. M.
Parichy
,
M. R.
Elizondo
,
M. G.
Mills
,
T. N.
Gordon
, and
R. E.
Engeszer
,
Dev. Dyn.
238
(
12
),
2975
3015
(
2009
).
20.
S.
Subendran
,
Y.-C.
Wang
,
Y.-H.
Lu
, and
C.-Y.
Chen
,
Sci. Rep.
11
(
1
),
13801
(
2021
).
21.
A.
Khalili
,
N.
Safarian
,
E.
van Wijngaarden
,
G. S.
Zoidl
,
G. R.
Zoidl
, and
P.
Rezai
,
J. Neurosci. Res.
101
(
12
),
1814
1825
(
2023
).
22.
A.
Nady
,
A. R.
Peimani
,
G.
Zoidl
, and
P.
Rezai
,
Lab Chip
17
(
23
),
4048
4058
(
2017
).
23.
A.
Khalili
,
E.
van Wijngaarden
,
K.
Youssef
,
G. R.
Zoidl
, and
P.
Rezai
,
Biotechnol. J.
17
(
1
),
2100076
(
2022
).
24.
X.
Lin
,
S.
Wang
,
X.
Yu
,
Z.
Liu
,
F.
Wang
,
W. T.
Li
,
S. H.
Cheng
,
Q.
Dai
, and
P.
Shi
,
Lab Chip
15
(
3
),
680
689
(
2015
).
25.
C.-Y.
Chen
,
C.-Y.
Chen
,
C.-Y.
Lin
, and
Y.-T.
Hu
,
Lab Chip
13
(
14
),
2834
2839
(
2013
).
26.
F.
Ellett
and
D.
Irimia
,
Zebrafish
14
(
2
),
140
145
(
2017
).
27.
D.
Loganathan
,
C.-L.
Hsieh
,
B.-E.
Shi
,
Y.-H.
Lu
, and
C.-Y.
Chen
,
Adv. Mater. Technol.
8
(
3
),
2201073
(
2023
).
28.
E.
Diller
and
M.
Sitti
,
Adv. Funct. Mater.
24
(
28
),
4397
4404
(
2014
).
29.
B.
Panigrahi
,
V.
Sahadevan
, and
C.-Y.
Chen
,
iScience
24
(
12
),
103367
(
2021
).
30.
D.
Loganathan
,
C.-Y.
Ou
,
C.-W.
Hsu
, and
C.-Y.
Chen
,
Adv. Mater. Technol.
9
,
2401135
(
2024
).
31.
N.
Okabe
,
B.
Xu
, and
R. D.
Burdine
,
Dev. Dyn.
237
(
12
),
3602
3612
(
2008
).
32.
S.
Subendran
,
C.-F.
Wang
,
D.
Loganathan
,
Y.-H.
Lu
, and
C.-Y.
Chen
,
Sci. Rep.
12
(
1
),
5041
(
2022
).
33.
D.
Loganathan
,
Y.
Chaung
,
Y.-H.
Lu
,
C.-H.
Cheng
, and
C.-Y.
Chen
,
Adv. Mater. Technol.
9
(
10
),
2400292
(
2024
).
34.
A.
Khalili
,
A. R.
Peimani
,
N.
Safarian
,
K.
Youssef
,
G.
Zoidl
, and
P.
Rezai
,
Integr. Biol.
11
(
10
),
373
383
(
2019
).
35.
C.-Y.
Chen
,
C.-Y.
Lin
, and
Y.-T.
Hu
,
Exp. Fluids
55
(
7
),
1765
(
2014
).
36.
K.
Mani
,
Y.-C.
Hsieh
,
B.
Panigrahi
, and
C.-Y.
Chen
,
Biomicrofluidics
12
(
2
), 021101 (
2018
).
37.
A.
Johnson
,
E.
Loh
,
R.
Verbitsky
,
J.
Slessor
,
B. C.
Franczak
,
M.
Schalomon
, and
T. J.
Hamilton
,
Sci. Rep.
13
(
1
),
3768
(
2023
).
38.
D.
Loganathan
,
S.-H.
Wu
, and
C.-Y.
Chen
,
Lab Chip
23
(
1
),
106
114
(
2022
).
39.
J. D.
Monroe
,
D. P.
Manning
,
P. M.
Uribe
,
A.
Bhandiwad
,
J. A.
Sisneros
,
M. E.
Smith
, and
A. B.
Coffin
,
Hear. Res.
341
,
220
231
(
2016
).
40.
Z.
Lu
and
A. A.
DeSmidt
,
J. Assoc. Res. Otolaryngol.
14
(
4
),
509
521
(
2013
).
41.
S.
Subendran
,
C.-W.
Kang
, and
C.-Y.
Chen
,
Micromachines
12
,
68
(
2021
).
42.
A. A.
Bhandiwad
,
D. G.
Zeddies
,
D. W.
Raible
,
E. W.
Rubel
, and
J. A.
Sisneros
,
J. Exp. Biol.
216
(
Pt 18
),
3504
3513
(
2013
).
43.
K.
Raj M
and
S.
Chakraborty
,
J. Appl. Polym. Sci.
137
(
27
),
48958
(
2020
).
44.
A. N.
Popper
,
A. D.
Hawkins
,
O.
Sand
, and
J. A.
Sisneros
,
J. Acoust. Soc. Am.
146
(
2
),
948
955
(
2019
).
45.
K.
Heise
,
H.
Yurk
,
C.
Nordstrom
, and
L.
Barrett-Lennard
,
Adv. Exp. Med. Biol.
875
,
455
459
(
2016
).
46.
M. E.
Halpern
,
J.
Rhee
,
M. G.
Goll
,
C. M.
Akitake
,
M.
Parsons
, and
S. D.
Leach
,
Zebrafish
5
(
2
),
97
110
(
2008
).
47.
S.
Cassar
,
I.
Adatto
,
J. L.
Freeman
,
J. T.
Gamse
,
I.
Iturria
,
C.
Lawrence
,
A.
Muriana
,
R. T.
Peterson
,
S.
Van Cruchten
, and
L. I.
Zon
,
Chem. Res. Toxicol.
33
(
1
),
95
118
(
2020
).
48.
K.
Mani
,
T. C.
Chang Chien
,
B.
Panigrahi
, and
C. Y.
Chen
,
Sci. Rep.
6
,
36385
(
2016
).
49.
B. D.
Fontana
,
M.
Cleal
,
A. J.
Gibbon
,
S. D.
McBride
, and
M. O.
Parker
,
Neuropharmacology
196
,
108681
(
2021
).
50.
B.
Panigrahi
and
C.-Y.
Chen
,
Lab Chip
19
(
24
),
4033
4042
(
2019
).
51.
Q.
Lu
,
D.
Tucker
,
Y.
Dong
,
N.
Zhao
, and
Q.
Zhang
,
Mol. Neurobiol.
53
(
8
),
5344
5355
(
2016
).
52.
W.-C.
Tian
and
E.
Finehout
,
Microfluidics for Biological Applications
(
Springer Science & Business Media
,
2009
).
53.
D.
Kobylarz
,
M.
Noga
,
A.
Frydrych
,
J.
Milan
,
A.
Morawiec
,
A.
Glaca
,
E.
Kucab
,
J.
Jastrzębska
,
K.
Jabłońska
,
K.
Łuc
,
G.
Zdeb
,
J.
Pasierb
,
J.
Toporowska-Kaźmierak
,
S.
Półchłopek
,
P.
Słoma
,
M.
Adamik
,
M.
Banasik
,
M.
Bartoszek
,
A.
Adamczyk
,
P.
Rędziniak
,
P.
Frączkiewicz
,
M.
Orczyk
,
M.
Orzechowska
,
P.
Tajchman
,
K.
Dziuba
,
R.
Pelczar
,
S.
Zima
,
Y.
Nyankovska
,
M.
Sowińska
,
W.
Pempuś
,
M.
Kubacka
,
J.
Popielska
,
P.
Brzezicki
, and
K.
Jurowski
,
Toxics
11
(
9
), 723 (
2023
).
54.
H.
Xue
,
A.
Thaivalappil
, and
K.
Cao
,
Cells
10
(
12
),
3379
(
2021
).
55.
S.
Li
,
Y.
Cui
,
M.
Wen
, and
G.
Ji
,
Toxics
11
, 594 (
2023
).
56.
G. D. P.
Cognato
,
J. W.
Bortolotto
,
A. R.
Blazina
,
R. R.
Christoff
,
D. R.
Lara
,
M. R.
Vianna
, and
C. D.
Bonan
,
Neurobiol. Learn. Memory
98
(
4
),
321
328
(
2012
).
57.
H.
Ikeda
,
A. H.
Delargy
,
T.
Yokogawa
,
J. M.
Urban
,
H. A.
Burgess
, and
F.
Ono
,
PLoS One
8
(
5
),
e63318
(
2013
).
You do not currently have access to this content.