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Neuroanatomical localization of galanin in zebrafish telencephalon and anticonvulsant effect of galanin overexpression. Piotr Podlasz, Anna Jakimiuk, Natalia Kasica-Jarosz, Krzysztof Czaja, and Krzysztof Wasowicz ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00239 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018
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ACS Chemical Neuroscience
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Neuroanatomical localization of galanin in zebrafish
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telencephalon and anticonvulsant effect of galanin overexpression.
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Piotr Podlasz1*, Anna Jakimiuk1, Natalia Kasica-Jarosz2, Krzysztof Czaja3, Krzysztof
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Wasowicz1.
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Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland.
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Mazury in Olsztyn, Poland.
Department of Pathophysiology, Forensic Veterinary and Administration, Faculty of
Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and
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Athens, GA, USA
Department of Veterinary Biosciences and Diagnostic Imaging, University of Georgia.
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Abstract
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Galanin is a neuropeptide widely expressed in the nervous system, but it is also
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present in nonneuronal locations. In the brain galanin may function as an inhibitory
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neurotransmitter. Several studies have showed that galanin is involved in seizure regulation
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and can modulate epileptic activity in the brain. The overall goal of the study was to establish
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zebrafish as a model to study the antiepileptic effect of galanin. The goal of this study was
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achieved by: 1) determining neuroanatomical localization of galanin in zebrafish lateral
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pallium, which is considered to be the zebrafish homologue of the mammalian hippocampus,
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the brain region essential for initiation of seizures; and 2) testing the anticonvulsant effect of
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galanin
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pentylenotetrazole(PTZ)-seizure model in larval zebrafish using automated analysis of motor
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function and qPCR were used in the study. Immunohistochemical staining of zebrafish larvae
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revealed numerous galanin-IR fibers innervating the subpallium, but only scarce fibers
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reaching the dorsal parts of telencephalon, including lateral pallium. In 3-month old zebrafish
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galanin-IR innervation of the telencephalon was similar, however much more galanin-IR
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fibers reached the dorsal telencephalon, but in the lateral pallium only scarce galanin-IR fibers
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were visible. qRT-PCR revealed, as expected, a strong increase in the expression of galanin in
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Tg(hsp70l:galn) line after heat shock, however also without heat shock the galanin expression
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was several-fold higher than in the control animals. Galanin overexpression resulted in
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downregulation of c-fos after PTZ treatment.
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overexpression inhibited locomotor activity in PTZ-treated and control larvae. The obtained
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results show that galanin overexpression reduced the incidence of seizure-like behavior
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episodes and their intensity but had no significant effect on their duration. The findings
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indicate that in addition to antiepileptic action, galanin modulates arousal behavior and
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demonstrates a sedative effect. The current study showed that galanin overexpression
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correlated with a potent anticonvulsant effect in the zebrafish PTZ seizure-model.
overexpression.
Whole
mount
immunofluorescence
and
Behavioral analysis showed that galanin
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staining
Keywords
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galanin, epilepsy, zebrafish, pentylenotetrazole, lateral pallium, seizures, c-fos,
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Introduction
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Galanin is a 29-30 amino acid long neuropeptide, which was first discovered in
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porcine intestine 1. Galanin is widely expressed in the central and peripheral nervous system
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in many studied species
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nonneuronal locations, including the skin, 13 and immune cells 14, 15. In the brain galanin may
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function as an inhibitory neuromodulator/neurotransmitter or as a hypophysiotropic
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messenger in the anterior pituitary. Neuroanatomical localization and physiological properties
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of galanin suggest that this peptide may be involved in the regulation of seizures. Galanin is
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highly expressed in the hippocampus, which is the major gateway in the propagation of
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seizure activity 16 and exerts a presynaptic inhibitory effect on the glutamatergic transmission,
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inhibiting epileptic seizures
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seizure regulation and can modulate epileptic activity in the brain
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including zebrafish
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- , but also has been found in
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. Several studies have showed that galanin is involved in 17, 18, 20
.
The zebrafish (Danio rerio) is a potential model organism for studying the
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mechanisms underlying animal and human disease and drug screening
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many zebrafish models of human diseases including epilepsy 24,
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number of features making them an attractive research tool. The zebrafish has a rapid
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generation rate (3–4 months) and the ability to generate clutch sizes of 50–200 embryos from
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a pair almost every week. The developing zebrafish is transparent, and the larval brain shows
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signs of an everted telencephalic organization by as early as 5 days post-fertilization (dpf). In
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the larval zebrafish brain, all of the major adult subdivisions are present, and the optic tectum
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begins to take a layered ‘cortical’ organization at 5 dpf. All cell types that are necessary for
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the generation of abnormal excitatory discharge within a network are present and functional
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- . There are
25, 26
. Zebrafish have a
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as early as in the 5 dpf zebrafish 26, 27, 28, 29. A fundamental advantage is that zebrafish share a
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considerable amount of genetic identity with humans, and several of their organ systems are
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remarkably similar to those found in mammals. The hippocampus is one of the major brain structures responsible for the propagation
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of seizure activity
. The hippocampus and, particularly, the dentate gyrus receives a rich
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innervation of galanin-containing fibers
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allocortex which is a phylogenetically older area of cerebral cortex in the telencephalon
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Most of the fish brain shows conserved similarities to the mammalian brain, however the fish
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telencephalon is very different from the mammalian counterpart, and neuroanatomical
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homologies remain uncertain. Currently, it is believed that the fish lateral pallium plays a
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similar role as the hippocampus of mammals 32, 33, 34, 35, 36, 37.
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. In mammals the hippocampus is located in the 31
.
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The overall goal of the study was to establish zebrafish as a model to study the
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antiepileptic effect of galanin. The goal was achieved by determining the neuroanatomical
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localization of galanin in zebrafish lateral pallium, which is considered as a zebrafish
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homologue of the mammalian hippocampus, representing the brain region essential for the
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initiation of seizures, and by testing the anticonvulsant effect of galanin overexpression, in a
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zebrafish pentylenotetrazole (PTZ)-seizure model.
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Results and Discussion
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Whole mount immunohistochemistry
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Immunohistochemical staining of 5 dpf (data not shown) and 7 dpf zebrafish larvae
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revealed
numerous galanin-IR fibers innervating the ventral part of the telencephalon
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(subpallium), but only scarce fibers reaching the dorsal pallial parts of telencephalon
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including lateral pallium, which is considered to be the zebrafish homologue of the
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mammalian hippocampus. Additionally, one numerous group of galanin-IR neurons was
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visible in this area. The galanin-IR neurons were located in the preoptic area (POA), posterior
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to anterior commissure (Cant). (Fig. 1, Supp. Video 1).
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Fig. 1. Whole-mount immunofluorescence staining of 7 dpf zebrafish brain using
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antibodies against galanin (green) and tyrosine hydroxylase (dopaminergic marker; red).
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Pictures were taken using a confocal microscope. Figures represent optical sections from the
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stack of images taken every 10 µm. For the whole stack of images see suppl. fig 1. Dotted
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yellow lines mark approximate location of the lateral pallium. Cant anterior commissure, 5 ACS Paragon Plus Environment
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Cpop supraoptic commissure, Ctub commissure of the posterior tuberculum, D dorsal
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telencephalic area, OB olfactory bulb, POA preoptic area, TeO optic tectum. Scale bar =100
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µm.
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In the 3-month old zebrafish, galanin-IR innervation of the telencephalon was similar
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to the larval one, however much more galanin-IR was found to reach the dorsal telencephalon.
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Numerous galanin-IR nerve fibers were observed in the central and medial zone of the dorsal
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telencephalic area. In the lateral zone of the dorsal telencephalic area, only scarce galanin-IR
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fibers were visible (Fig. 2, Supp. Video 2).
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Fig 2. Whole-mount immunofluorescence staining of 3-month-old zebrafish forebrain using
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antibodies against galanin (green) and neuronal marker Hu (red). Pictures were taken using a
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confocal microscope. Figures represent optical sections from the stack of images taken every
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24 µm. For the whole stack of images see suppl. fig. 2. Dotted yellow lines mark approximate
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location of the lateral pallium. There were only scarce galanin-IR fibers found innervating this
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area of the adult zebrafish brain. Dc central zone of dorsal telencephalic area, Dl lateral zone
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of dorsal telencephalic area, Dm medial zone of dorsal telencephalic area, OB olfactory bulb,
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POA preoptic area. Scale bar =100 µm.
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qRT-PCR
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qRT-PCR was performed to determine the expression level of galanin and to
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investigate if its level correlates with the mRNA level of c-fos (marker of neuronal activity) in
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the PTZ seizure model (Fig. 3). As expected after heat shock the mRNA level of galanin in the
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transgenic line Tg(hsp70l:galn) dramatically increased (over 300 fold, p value
