Expression of the eight GABAA receptor α subunits in the developing zebrafish central nervous system

GABA is a robust regulator of both developing and mature neural networks. It exerts many of its effects through GABAA receptors, which are heteropentamers assembled from a large array of subunits encoded by distinct genes. In mammals, there are 19 different GABAA subunit types, which are divided into the α, β, γ, δ, ε, π, θ and ρ subfamilies. The immense diversity of GABAA receptors is not fully understood. However, it is known that specific isoforms, with their distinct biophysical properties and expression profiles, tune responses to GABA. Although larval zebrafish are well-established as a model system for neural circuit analysis, little is known about GABAA receptors diversity and expression in this system. Here, using database analysis, we show that the zebrafish genome contains at least 23 subunits. All but the mammalian θ and ε subunits have at least one zebrafish ortholog, while five mammalian GABAA receptor subunits have two zebrafish orthologs. Zebrafish contain one subunit, β4, which does not have a clear mammalian ortholog. Similar to mammalian GABAA receptors, the zebrafish α subfamily is the largest and most diverse of the subfamilies. In zebrafish there are eight α subunits, and RNA in situ hybridization across early zebrafish development revealed that they demonstrate distinct patterns of expression in the brain, spinal cord, and retina. Some subunits were very broadly distributed, whereas others were restricted to small populations of cells. Subunit-specific expression patterns in zebrafish resembled were those found in frogs and rodents, which suggests that the roles of different GABAA receptor isoforms are largely conserved among vertebrates. This study provides a platform to examine isoform specific roles of GABAA receptors within zebrafish neural circuits and it highlights the potential of this system to better understand the remarkable heterogeneity of GABAA receptors.

(previously referred to as GABA c receptors) [4,5]. Each subunit is encoded by a discrete gene 54 that is spatially and developmentally regulated to generate distinct, but often overlapping, 55 expression patterns [6][7][8][9]. Alternative splicing and RNA editing of some subunits further 56 increases the number of subtypes available [10]. Although this extensive receptor heterogeneity 57 is not fully understood, some subunits confer distinct biophysical and pharmacological 58 properties, interact with specific cytoplasmic proteins, and localize to specific subcellular 59 domains [11][12][13]. Ultimately, this receptor diversity provides a capacity to tailor responses to 60 GABA within neural circuits.  [23], and the Mauthner cells, a pair of well-studied reticulospinal neurons found in the amphibian and teleost hindbrain [24]. In these studies, the GABA A receptor isoforms were 93 not identified. However, patch-clamp recordings of the Mauthner cells showed three distinct 94 GABA A kinetic profiles, which were proposed to be caused by different receptor isoforms [25].

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There is limited information about the heterogeneity of GABA A receptors in zebrafish.

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The most extensive study to date identified 23 GABA A receptor subunits and examined their  125

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Zebrafish were raised and maintained using established husbandry procedures.

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Embryos were kept at 28.5 °C in E3 media and staged according to morphological criteria [33].

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All experiments were performed using Tuebingen (Tu) or tub longfin (TLF) wild type embryos.

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To establish the number and organization of zebrafish GABA A receptor subunits, we used 182 mouse GABA A receptor subunit amino acid sequences to query zebrafish genome databases.

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We identified 23 zebrafish GABA A receptor subunits, each encoded by a distinct gene.

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Frequently splice variants were observed. In these cases, the principle splice isoform, as 185 indicated by the databases, was selected for analysis. 12 of the 19 mouse GABA A receptor 186 subunits were found to have a single ortholog in zebrafish (Fig 1). Amino acid identities between 187 mouse and zebrafish orthologs ranged from 53.1 to 86.3%. Five additional mouse subunits,

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Expression of gabra1, gabra2a, gabra2b, gabra3, gabra4 and gabra5 was detected at 24 229 hpf (Fig 2). Neither gabra6a or gabra6b were detected at this developmental stage. gabra2a 230 and gabra2b were detected broadly and did not appear to be spatially restricted (Fig 2C-F). To 231 distinguish whether these gene transcripts were widespread or background due to the probe, a 232 second probe was generated for each gene (S1 Table). These probes yielded widespread 233 staining, very similar to the initial probes used, which suggests that gabra2a and gabra2b are 234 widely distributed. Consistent with these findings, a previous study reported that gabra2a 235 exhibits a diffuse, broad pattern of expression in larval zebrafish [28].  (Fig 2A, B). Although expressed in discrete cells, gabra3 was widely expressed, and 249 found within the tegmentum, hindbrain, and ventral and intermediate domains in the spinal cord 250 (Fig 2G, H). gabra4 was expressed in a diffuse manner throughout the brain but specifically 251 within a select population of ventral spinal cord cells (Fig 2I, J). The location and distribution of 252 these cells suggests that gabra4 is expressed selectively within Kolmer-Agduhr (KA) neurons.

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The expression of all GABA A receptor subunit genes, except for gabra6a, was observed 257 at 48 hpf (Fig 3). Similar to the pattern observed at 24 hpf, the gabra2 paralogs were again 258 detected broadly, with little spatial restriction (Fig 3C-F). Similar to its expression at 24hpf, 259 gabra1 is prominently expressed in the olfactory bulbs, the pallium and discrete clusters of cells 260 in medial portions of the medulla (Fig 3A, B). gabra3 was observed in relatively small clusters of 261 cells in the pallium, thalamus, and the medulla (Fig 3G, H).

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Transcripts for all α subunits are detected in larval zebrafish at 96 hpf (Fig 4). As with the 282 earlier developmental time points, gabra2a and gabra2b transcripts were detected broadly, 283 although gabra2b appears less diffuse and more discrete compared to at 24 and 48 hpf (Fig 4C-284 F). gabra1, gabra3, and gabra6b are also expressed more broadly compared to earlier stages.

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In contrast, gabra4, gabra5 and gabra6a transcripts were detected in smaller groups of cells.
gabra4 is expressed in a stripped pattern in the outer nuclear layer of the retina, the posterior 287 tuberculum area, which is a portion of the diencephalon, and the tectum, cerebellum, and 288 medulla (Fig I, J). gabra5 is expressed in discrete cells in the pallium, hypothalamus, cerebellum 289 and medulla (Fig K, L). Within the medulla, gabra5 expression in the Mauthner Cells is robust, 290 as it is at earlier developmental stages.