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/lp/springer-journals/the-glycine-receptor-alpha-3-subunit-mrna-expression-shows-sex-Cch50lr4La
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- Springer Journals
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- Copyright © The Author(s) 2023
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- 10.1186/s12868-023-00800-9
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Abstract
Background The glycinergic system plays an important inhibitory role in the mouse central nervous system, where glycine controls the excitability of spinal itch- and pain-mediating neurons. Impairments of the glycine receptors can cause motor and sensory deficits. Glycine exerts inhibition through interaction with ligand-gated ion channels com- posed of alpha and beta subunits. We have investigated the mRNA expression of the glycine receptor alpha 3 (Glra3) subunit in the nervous system as well as in several peripheral organs of female and male mice. Results Single-cell RNA sequencing (scRNA-seq) data analysis on the Zeisel et al. (2018) dataset indicated widespread but low expression of Glra3 in vesicular glutamate transporter 2 (Vglut2, Slc17a6) positive and vesicular inhibitory amino acid transporter (Viaat, Slc32a1)positive neurons of the mouse central nervous system. Highest occurrence of Glra3 expression was identified in the cortex, amygdala, and striatal regions, as well as in the hypothalamus, brainstem and spinal cord. Bulk quantitative real-time-PCR (qRT-PCR) analysis demonstrated Glra3 expression in cortex, amyg- dala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Addi- tionally, male mice expressed higher levels of Glra3 in all investigated brain areas compared with female mice. Lastly, RNAscope spatially validated Glra3 expression in the areas indicated by the single-cell and bulk analyses. Moreover, RNAscope analysis confirmed co-localization of Glra3 with Slc17a6 or Slc32a1 in the central nervous system areas sug- gested from the single-cell data. Conclusions Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent dif- ferences have been identified, indicating higher levels of Glra3 in several telencephalic and diencephalic areas, as well as in cerebellum and brainstem, in male mice compared with female mice. Keywords Glycine, Glra3, Brain, Spinal cord, Mice, Sex-dependent differences Introduction The amino acid glycine acts as an inhibitory neurotrans - *Correspondence: mitter in mammals and contributes to the regulation of Malin C. Lagerström both itch- and pain-associated networks [1, 2]. Glycine Malin.Lagerstrom@igp.uu.se is an agonist to the glycine receptors (GlyRs), which are Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden pentameric ligand-gated ion channels that predomi- Department of Anatomy, Physiology and Biochemistry, Swedish nantly consist of four ligand binding alpha (α) subunits University of Agricultural Sciences, Uppsala, Sweden (GLRA1–GLRA4) and one structural beta (β) subunit [3]. Neuropharmacology and Addiction, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden The Glra3 gene was first cloned from a rat brain cDNA library by homology screening, and found to be expressed © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ceder et al. BMC Neuroscience (2023) 24:32 Page 2 of 17 in the spinal cord [4]. Rat Glra3 mRNA has also been peripheral nervous system, its expression alongside located in sub-regions of the olfactory bulb, cerebral cor- expression of the excitatory marker Slc17a6 (Vglut2) tex, thalamus, hippocampus, cerebellum [5], and the retina and the inhibitory marker Slc32a1 (Viaat) were exam- [6]. Analysis in mice has mapped the Glra3 gene to chro- ined in neuronal cells from distinct nervous system mosome 8 [7], and transcriptional analyses of the spinal areas in the Zeisel et al. (2018) scRNA-seq dataset cord and dorsal root ganglia (DRG) have located Glra3 [22]. Glra3 in the central and peripheral nervous sys- expression to the spinal dorsal horn. In contrast, expres- tem showed low levels of expression (as indicated by sion of Glra3 was not detected in DRG and below detec- the light-blue colored dots) and was found to be aber- tion level in the ventral spinal horn [8]. Additionally, the rantly expressed throughout cells of a given group (as Allen Institute has mapped Glra3 to both the dorsal and indicated by the size of the dots’ diameter) (Fig. 1A, ventral horn as well as to different divisions of the spinal Table 1). In the brain, the highest occurrence (> 1.0%) cord (https:// mouse spinal. brain- map. org/ image series/ of Glra3 expression was found in the amygdala (3.0% detail/ 10002 9493. html) [9]. Immunohistochemical analysis expressed Glra3) (Fig. 1A, Table 1). Said brain region of GLRA3 expression in the mouse spinal cord has located showed a small overlap between Glra3 and Slc17a6 the subunit’s expression to the dorsal horn [10, 11]. Func- expression, but no expression of Slc32a1 was detected tionally, spinal GLRA3 has a role in certain inflammatory (Fig. 1B). Other telencephalic areas with more than pain states, where ablation or mutations of Glra3 results in 1.0% of neurons expressing Glra3 were the cortex and faster hypersensitivity recovery [10, 12, 13]. some striatal regions. Expression of Glra3 was less than Although GLRA3 is mainly known for its role in spinal 0.5% in the olfactory bulb, striatum ventral, hippocam- circuits, the subunit is also expressed in the developing and pus, and dentate gyrus (Fig. 1A, Table 1). In the cortex, adult brain [14], and a connection between GLRA3, etha- the Glra3-expressing cells displayed low co-localization nol-mediated effects [15, 16] as well as respiratory rhyth- with Slc17a6 and no overlap with Slc32a1. In contrast, mic activity [17] has been reported. Immunohistochemical in the striatum, both Glra3 and Slc32a1 were expressed, and electrophysiological analyses in mice have localized but no expression of Slc17a6 was observed (Fig. 1B). GLRA3 to post-synaptic sites in the inner plexiform layer In the diencephalon, Glra3 expression was detected in of the retina [18], the nucleus accumbens [15, 19], dorsal the hypothalamus (1.1% of the neurons expressed the striatum and medial prefrontal cortex pyramidal neurons targeted gene in this area), where both inhibitory and of layer II/III [19], as well as to both presynaptic glycine excitatory neurons displayed some levels of expression transporter 2 (GLYT2)-expressing neurons and postsyn- (Fig. 1B). Expression of Glra3 (0.4%) was also found aptic neurons in brainstem areas important for respiratory in thalamic neurons, but mainly in excitatory neurons rhythms [17]. In situ hybridization also locates Glra3 to the (Fig. 1B). In the brainstem, Glra3 was found in the dor- cortex, hypothalamus and midbrain (https:// mouse. brain- sal, dorsal–ventral and ventral midbrain neurons, with map. org/ exper iment/ show/ 70723 453) [20]. Moreover, the dorsal–ventral midbrain Glra3 neurons being Slc32a1- expression of Glra3 and immunohistochemical detection expressing, and dorsal and ventral midbrain Glra3 neu- of GLRA3 are increased in the insular cortex of female rons being mainly Slc17a6-expressing (dorsal midbrain: mice in an endometriosis model [21]. 3.5%, dorsal–ventral midbrain: 1.3%; ventral midbrain: The cited studies suggest that GLRA3 is expressed in 1.9% of neurons expressed Glra3) (Fig. 1B, Table 1). several areas in the central nervous system. However, a Glra3 was detected in 1.5% of the pons neurons and detailed analysis of the Glra3 mRNA expression, using 3.4% of the medulla neurons (Fig. 1A, Table 1). In both complementary methods, in both sexes of mice is lacking. areas, Glra3-positive neurons showed partial over- We have therefore investigated the expression of Glra3 lap with Slc17a6 and Slc32a1 expression (Fig. 1B). in adult female and male mice using quantitative real- Expression of Glra3 was not detected in the cerebel- time-PCR (qRT-PCR) and a sensitive fluorescent in situ lum (Fig. 1A). In the spinal cord, 3.2% of neurons were hybridization method called RNAscope. Furthermore, we Glra3-positive and co-expression with both Slc17a6 compared our findings with a publicly available single-cell and Slc32a1 was identified. In the peripheral nervous RNA sequencing (scRNA-seq) dataset. system, Glra3 expression was detected in one neuron of the enteric nervous system (0.1%), and not detected Results in the DRG, as described in previous studies [8, 10, 11], Glra3 exhibited low expression in excitatory and inhibitory nor in sympathetic ganglion neurons (Fig. 1A, B). neurons in several areas of the central nervous system In conclusion, the scRNA-seq analysis of the Zeisel Expression of Glra3 mRNA can be detected in several et al. (2018) dataset revealed that Glra3 was expressed areas of the brain and spinal cord [4, 5, 8]. To further in low levels in both excitatory and inhibitory neurons investigate the expression of Glra3 in the central and in several brain areas, as well as in the spinal cord. For C eder et al. BMC Neuroscience (2023) 24:32 Page 3 of 17 Fig. 1 Low expression of Glra3 was detected in the central nervous system. The expression of Glra3 and its co-expression with excitatory marker Slc17a6 (Vglut2) and inhibitory marker Slc32a1 (Viaat) were examined in distinct areas in the central and peripheral nervous system in the Zeisel et al. (2018) dataset, which contained scRNA-seq data of 27,998 genes in 74,539 neurons [22]. A Dot plot of the expressions of the targeted genes in all neurons in the areas annotated in the Zeisel et al. (2018) dataset. Glra3 was generally expressed in low levels, as indicated by light blue colored dots, and in a small number of cells, as indicated by the dots’ small diameters. The highest expression was found in Zeisel et al. (2018) defined central nervous system areas, namely the amygdala, dorsal midbrain, medulla and the spinal cord (more than 3.0% of neurons in these areas expressed Glra3). B The occurrence of Glra3 neurons (Glra3 was considered expressed if log1p > 0.1) and the co-expression of Slc17a6 and Slc32a1 in the respective areas visualized with a dot plot. Glra3 was expressed in both excitatory and inhibitory neurons, with some areas displaying either excitatory or inhibitory Glra3 neurons, while other areas contained Glra3 neurons of both molecular properties. Ceder et al. BMC Neuroscience (2023) 24:32 Page 4 of 17 Table 1 Expression of Glra3 in the distinct nervous system areas defined in the Zeisel et al. (2018) single-cell RNA sequencing dataset Nervous system area Total number of cells Number of Glra3 expressing Relative abundance of cells* Glra3 expression in area (%) Olfactory bulb 7763 4 0.1 Cortex 15,205 176 1.2 Amygdala 956 29 3.0 Pallidum 832 5 0.6 Striatum dorsal 1962 ND ND Striatum dorsal–ventral, Amygdala 121 2 1.7 Striatum dorsal–ventral 1332 14 1.1 Striatum ventral 3676 14 0.4 Striatum dorsal–ventral, Dentate gyrus 933 ND ND Hypothalamus 1981 21 1.1 Thalamus 3029 13 0.4 Hippocampus, Cortex 7507 20 0.3 Hippocampus 3210 7 0.2 Dentate gyrus 6177 1 0.01 Midbrain dorsal 5204 181 3.5 Midbrain dorsal–ventral 460 6 1.3 Midbrain ventral 313 6 1.9 Pons, Medullae, Cerebellum 158 ND ND Cerebellum 3240 ND ND Pons 1196 18 1.5 Medulla 1566 53 3.4 Spinal Cord 1790 57 3.2 Sympathetic ganglion 886 ND ND Dorsal root ganglion 1580 ND ND Enteric nervous system 943 1 0.1 ND not detected. *Glra3 considered expressed if log1p < 0.1 more detailed information about the expression pattern visceral organs, namely the heart and spleen, for both of Glra3, see Table 1. females and males, as well as the lung, kidney and tes- tis tissues for male mice only. However, the measured Ct values of Glra3 in the heart, lung, kidney, spleen Glra3 mRNA expression was mainly found in the central and testes should be carefully reviewed, as Glra3 was nervous system difficult to measure in the visceral organs compared Analysis on the single-cell level revealed that Glra3 with the tissues collected from the central nervous sys- was predominantly expressed in the cortex, amyg- tem. The relative fluorescence unit (RFU) was low, and dala, striatum, hypothalamus, brainstem and spinal many cycles (40–45 cycles) were needed to obtain a cord, where Glra3 was expressed in more than 1.0% of Ct-value, making it difficult to separate the amplifica- the neurons in each area. To broaden the analysis, we tion of Glra3 in the visceral organs from background studied the relative mRNA expression of Glra3 in both noise and non-specific amplification (Additional file 1: sexes of C57BL/6J mice using qRT-PCR. Glra3 was Fig. S1). Compared with females, males generally had expressed widespread in the central nervous system higher expression of Glra3 in the cortex (p = 0.0256), in both females and males(Additional file 1: Table S1). amygdala (p = 0.0009), striatum (p = 0.0118), hypothal- In females, highest Glra3 expression levels were iden- amus (p = 0.0144), thalamus (p = 0.0317), hippocam- tified in the amygdala, hypothalamus, thalamus and pus [p = 0.0051 (without outlier), p = 0.0079 (with spinal cord (top 4 areas). In comparison, male mice outlier)], cerebellum [p = 0.0027 (without outlier), showed highest Glra3 expression levels in the amyg- p = 0.0079 (with outlier)] and brainstem (p = 0.0010), dala, hypothalamus, thalamus and brainstem (Fig. 2). but not in the pituitary gland (p > 0.9999) and spinal Furthermore, raw cycle threshold (Ct) values (indi- cord (p = 0.1495) (Fig . 2). In conclusion, male mice cating expression) of Glra3 could be detected in a few C eder et al. BMC Neuroscience (2023) 24:32 Page 5 of 17 Fig. 2 qRT-PCR revealed Glra3 to be mainly found in the central nervous system. Glra3 expression in adult female (n = 5, red) and adult male (n = 5, blue) C57BL/6J mice was measured using qRT-PCR, with a cutoff of 45 cycles. The relative mRNA expression was calculated using the delta Ct method with three stable reference genes (female and male body: Actβ, Rpl19, Gapdh; female brain: Actβ, Rpl19, Gapdh; male brain: Actβ, Rpl19, Cyclo). Stable reference genes were found using the GeNorm protocol [23]. Biological outliers (in total two outliers from male mice, one for the hippocampus and one for the cerebellum) were removed using the Grubbs outlier test with α = 0.05 before proceeding. The log2 fold mean difference (± SEM) against the genomic Glra3 DNA expression is illustrated in the combined scatter-bar plot. Glra3 was measured in heart (n = 3), lung (n = 3–5), spleen (n = 4–5), kidney (n = 4–5) and testes (n = 5) however these findings should be considered with caution (see Additional file 1: Fig. S1). Glra3 was expressed in most tissues collected for the central nervous system, with highest expression in the amygdala, hypothalamus, thalamus, brainstem and spinal cord. Two-tailed Mann–Whitney U-test (thalamus p = 0.0317; hippocampus p = 0.0079 (with outlier); cerebellum p = 0.0079 (with outlier); pituitary gland p > 0.9999) or unpaired t-test (prefrontal cortex p = 0.0256; amygdala p = 0.0009; striatum (females caudate putamen, males caudate putamen and nucleus accumbens); p = 0.0118; hypothalamus p = 0.0144; hippocampus p = 0.0051 (without outlier); cerebellum p = 0.0027 (without outlier); brainstem p = 0.0010; spinal cord p = 0.1495) were used to calculate the difference between female and male mice for each tissue where *p < 0.05, **p < 0.01, ***p < 0.001 displayed higher Glra3 mRNA levels compared with of Glra3 was detected in striatum of male mice, but not female mice in all brain areas, whereas peripheral in females (Additional file 1: Fig. S2B). No Glra3 probe organs expressed low to no expression of Glra3 regard- signal could be detected in cerebellum regardless of sex less of sex. (Additional file 1: Figure S2C). In all areas, except for the hippocampus, Glra3 co-expressed with Slc17a6, while Glra3 spatial analysis corroborated with the qRT‑PCR co-localization with Slc32a1 was found in all brain areas analysis except for the female thalamus (Figs. 3a1–c1, 4a1–c1, Following Glra3 detection in the cortex, amygdala, stria- Additional file 1: Fig. S2). tum, hypothalamus, thalamus, hippocampus, brainstem In the spinal cord of both females and males (Figs. 5, 6), and spinal cord based on single-cell and bulk analy- Glra3 was found in the dorsal horn of cervical (Figs. 5A, ses, with the latter analysis revealing a sex-dependent 6A), thoracic (Figs. 5B, 6B), lumbar (Figs. 5C, 6C) and expression pattern in several brain areas, we sought to sacral (Figs. 5D, 6D) divisions, with co-expressions of investigate the spatial Glra3 expression in these areas. Slc17a6 and Slc32a1 detected in all divisions. Lastly, in the cervical, lumbar and sacral divisions, Glra3 expres The analysis was conducted using fluorescent in situ - hybridization with the RNAscope approach [24] in sion was detected in the ventral horn (Figs. 5C, D, 6C, D). female and male C57BL/6J mice, with the aim of verify- In conclusion, spatial validations of Glra3 expression ver- ing the expression of Glra3. The analysis was not set out ified that Glra3 is expressed in all areas identified from to quantify Glra3 expression and thus, no expression the single-cell and bulk analyses. Co-localization of Glra3 level comparison was performed between females and with Slc17a6 was identified in all brain and spinal cord males. In both female and male brains (Figs. 3, 4), expres- areas, except for the hippocampus, and with Slc32a1 in sion of Glra3 was found in the cortex (Figs. 3A-a1, 4A-a1, all areas, except for the female thalamus. somatosensory cortex displayed in the image), amygdala (Figs. 3B-b1, 4B-b1), pallidum (Additional file 1: Fig. S2A Discussion and S2a1-3), hypothalamus (Figs. 3B-b2,4B-b2), thalamus Using three different mRNA-based methods, we here (Figs. 3B-b3, 4B-b3), hippocampus (Figs. 3B-b4, 4B-b4) report that the glycinergic receptor unit Glra3 is and brainstem (Figs. 3C-c1, 4C-c1) areas. Expression expressed in central nervous system areas such as the Ceder et al. BMC Neuroscience (2023) 24:32 Page 6 of 17 cortex, amygdala, striatum, hypothalamus, thalamus, [19, 25, 28] and the brainstem [17, 29]. For instance, hippocampus, brainstem, and spinal cord. Furthermore, McCracken et al. (2017) showed that GLRA3-contain- we identified that male mice display higher levels of ing GlyRs are found in various areas of the forebrain. Glra3 in the above listed areas, with the exception of the Additionally, when performing whole-cell recordings on −/− spinal cord. In all central nervous system areas, except Glra3 mice, it was reported that these mice lacked for the hippocampus, Glra3 expression overlapped with tonic inhibition in the forebrain. These findings indicate Slc17a6 expression, whereas co-expression of Glra3 and that Glra3 participates in tonic inhibition in the prefron- Slc32a1 was found in all of the targeted areas except for tal cortex and in both the dorsal striatum and nucleus the female thalamus. accumbens [19]. San Martin et al. (2021) investigated the potential role of the GLRA3 subunit in ethanol sen- Glra3 is expressed in several areas in the central nervous sitivity by focusing on the nucleus accumbens [15]. They system concluded that GLRA3 is expressed in low levels in the In the brain, GLRA3 exists as two isomers, namely the mouse nucleus accumbens [15]. Our qRT-PCR analy- shorter GLRA3K and the longer GLRA3L (additional 8A sis detected Glra3 in both the cortex and striatum, with exon), with the latter being the dominant variant in the higher levels found in male mice compared with female mouse brain [25]. In our analyses, the Glra3 qRT-PCR mice. Importantly, the dissected female striatum samples primers and the RNAscope probes targeted the nucleo- only contained caudate putamen, while the male striatum tide sequence outside the splicing area (primers: exon samples contained both caudate putamen and nucleus 9–10; probes: exon 1–8), meaning our analyses captured accumbens. This discrepancy could explain the difference the expression of both isomers. in expression levels in male and female striatum. How- The Human Protein Atlas project has mapped human ever, our RNAscope analysis identified a few Glra3 posi - GLRA3 [26] and mouse Glra3 [27] (https:// www. prote tive cells in males but none in females, suggesting that inatl as. org/ ENSG0 00001 45451- GLRA3/ brain) to several Glra3 expression may show a sex-dependent difference in areas in the central nervous system. In humans, GLRA3 striatum. has been detected in the cerebral cortex, amygdala, Earlier findings reported by Eichler et al. (2009) dem - hypothalamus, thalamus, hippocampal formation, mid- onstrate that the expression of the GLRA3L splice variant brain, basal ganglia and the brainstem (pons and medulla is dominant in mice [25], but in temporal lobe epilepsy, oblongata) [26]. In mice, the Human Protein Atlas pro- the shorter splice variant (GLRA3K) was upregulated. ject could locate Glra3 to the olfactory bulb, cerebral Through these findings, Eichler et al. (2009) concluded cortex, amygdala, hypothalamus, thalamus, hippocampal that both splice variants are located on glutamatergic formation, midbrain, basal ganglia, brainstem (pons and (3L) and GABAergic (3K) synaptic terminals [25]. Two medulla) and the cerebellum [27], similar to the expres- of our transcriptional analyses, where the excitatory and sion pattern displayed in the Allen Mouse Brain Atlas inhibitory characteristics were examined, also disclosed ( h t t p s :// mou s e. brain- ma p. or g/ e x p er imen t/ s how/ 73788 that Glra3 co-expresses with both an excitatory and an 474). These previous findings are coherent with our inhibitory marker. Schaefermeier and Heinze (2017) have results (Table 2). also reported expression of murine Glra3 in the hip- Previous GLRA3 studies have mainly focused on what pocampus [28], in a similar expression pattern as was function the subunit has in the brain, i.e. in the cortex observed herein in female and male mice (Figs. 3 and 4). [19], striatum, nucleus accumbens [15, 19], hippocampus (See figure on next page.) Fig. 3 Analysis of the spatial expression of Glra3 in the female brain. The spatial Glra3 mRNA expression was detected with RNAscope using probes against Glra3 (teal), Slc17a6 (red) and Slc32a1 (red). Expression was detected at (A) Bregma 0.98 mm in the (a1) cortex, and both co-localization of Glra3 with Slc17a6 or Slc32a1 was observed. B At Bregma -1.34 mm, Glra3 expression was found in the (b1) amygdala, (b2) hypothalamus, (b3) thalamus and (b4) hippocampus. b1–b4 Overlap with Slc17a6 expression was observed in all areas, except in the hippocampus, while overlap with Slc32a1 was seen in all areas, except in the thalamus. C At Bregma -6.84 mm, Glra3 expression and overlap with Slc17a6 or Slc32a1 expression was observed in the (c1) brainstem. Illustrations of the Bregma sections in A–C are adapted from https:// mouse. brain- map. org/ exper iment/ thumb nails/ 10004 8576? image_ type= atlas. The red dashed squares in A–C indicate approximately the area displayed in a1–c1. The dashed squares were consistently placed on the right side of the schematic image in order to maximize the readability of the abbreviations, regardless of the position of the representative images. White arrows denote examples of co-expression. Scale bars: 200 µm, enlargements 100 µm. Abbreviated areas in a1–c1: Aco = anterior cortical amygdaloid nucleus, AHP = anterior hypothalamic area, posterior part, BLA = basolateral amygdaloid nucleus, anterior part, BMA = basomedial amygdaloid nucleus, anterior part, DG = dentate gyrus, DM = dorsomedial hypothalamic nucleus, Gi = gigantocellular reticular nucleus, ME = medial amygdaloid nucleus, PLCo = posterolateral cortical amygdaloid nucleus, Rt = reticular thalamic nucleus, S1FL = primary somatosensory cortex, forelimb region, S1J = primary somatosensory cortex, jaw region, Sol = solitary tract, VL = ventrolateral thalamic nucleus, VMH = ventromedial hypothalamic nucleus, VPL = ventral posterolateral thalamic nucleus, VPM = ventral posteromedial thalamic nucleus. For all other abbreviations, please see https:// mouse. brain- map. org/ exper iment/ thumb nails/ 10004 8576? image_ type= atlas C eder et al. BMC Neuroscience (2023) 24:32 Page 7 of 17 Fig. 3 (See legend on previous page.) All our transcriptional Glra3 analyses mapped expres- subunit has also been suggested to have a potential sion in the caudal brainstem (medulla and pons). This is mechanism in mediating the presynaptic modulation of consistent with previous immunostainings performed by glycine release in the hypoglossal nucleus [29]. Manzke et al. (2010), in which ubiquitous GLRA3 expres- sion was detected in the brainstem [17]. The GLRA3 Ceder et al. BMC Neuroscience (2023) 24:32 Page 8 of 17 Glra3 is widely expressed in the spinal cord difficult to detect using qRT-PCR compared with tis - Previous PCR expressional analysis of Glra3 in the spi- sues collected from the central nervous system. The RFU nal cord reported that the gene is detected in the dorsal, was low, unstable amplification and melting curves were but not in the ventral spinal horn [8]. In addition to the obtained, and many cycles (40–45 cycles) were needed dense expression of Glra3 seen in the dorsal horn, our to obtain a Ct-value, making it difficult to separate the RNAscope analysis revealed that Glra3 was also detected proper amplification of Glra3 in the visceral organs from in the ventral (with a majority medioventrally) horns of background noise and non-specific amplification (e.g. the cervical, lumbar and sacral divisions. Therefore, a primer-dimer). As a result, we cannot confidently con - broader expression pattern of the Glra3 gene was dis- clude that Glra3 is expressed in these visceral organs. played when compared to an earlier report [8]. In Gro- Furthermore, chemical contamination, cycle-to-cycle emer et al. (2022), the division of the spinal cord that variability and random noise are systematic errors that was being analyzed was unspecified, suggesting that have been reported to affect results obtained with qRT- the ventral Glra3 expression might have been missed. PCR [32]. These interferences could possibly explain the Expression of Glra3 in the ventral horn has been shown variability in detection levels in some of the tested tis- by the Allen Institute, which is consistent with our find - sues. However, our findings in mice are reasonably con - ings. The spatial Glra3 analyses also showed that Glra3 sistent with what the Human Protein Atlas project and overlaps with sub-populations expressing Slc17a6 or Genotype-Tissue Expression project have reported on Slc32a1. Using the Zeisel et al. (2018) and Häring et al. the GLRA3 mRNA in humans [26]. The Human Protein (2018) datasets [22, 30], we also found Glra3 expres- Atlas project reports low levels of GLRA3 mRNA in adre- sion in both the excitatory SCGLU10 and Glut9, as well nal gland, pancreas, testes, female breast tissue, smooth as the inhibitory Gaba8-9 clusters, further demonstrat- muscle tissue, thymus, lymph nodes and tonsil tissue. ing the broad expression pattern of Glra3. Other studies Meanwhile the Genotype-Tissue Expression project have instead investigated GLRA3 expression in the spinal reports GLRA3 expression in the small intestine, testes cord using immunostaining [12, 13], where its detection and female breast tissue in 20–69 years old females and was restricted to the dorsal horn. The differences in the males using RNA sequencing (https:// www. prote inatl subunit’s protein expression, exhibited by immunostain- as. org/ ENSG0 00001 45451- GLRA3/ tissue), indicating ing, compared to our RNAscope analysis may have been inconsistencies in mRNA levels in the visceral organs. a result of not all Glra3 units being translated into pro- This inconsistency could be due to low levels of Glra3, tein [31]. the rate of mRNA turnover, or the point of transcription in which the tissues were harvested [33]. Therefore, what Glra3 expression in visceral organs role Glra3 has in visceral organs remains unknown. In this study, the Glra3 expression in visceral organs was investigated with bulk qRT-PCR. Raw Ct-values (indicat- Methodological considerations ing expression) of Glra3 could be detected in a few vis- In Zeisel et al. [22] the specific regions included in what ceral organs, namely the heart, lung, spleen, kidney and was labelled as cortex were not clearly specified, mak - testes. However, Glra3 expression in these organs was ing it unclear if the entire cortex was included or merely (See figure on next page.) Fig. 4 Analysis of the spatial expression of Glra3 in the male brain. The spatial Glra3 mRNA expression was detected with RNAscope using probes against Glra3 (teal), Slc17a6 (red) and Slc32a1 (red). Expression was detected at (A) Bregma 0.98 mm in the (a1) cortex, and both co-localization of Glra3 with Slc17a6 or Slc32a1 was observed. B At Bregma -1.34 mm, Glra3 expression was found in the (b1) amygdala, (b2) hypothalamus, (b3) thalamus and the (b4) hippocampus (for Slc32a1 the representative image is from a section between Bregma -1.06 and -1.22 mm). b1–b4 Overlap with Slc17a6 expression was observed in all areas, except for the hippocampus, while overlap with Slc32a1 expression was seen in all areas. C At Bregma -6.84 mm Glra3 expression and overlap with Slc17a6 or Slc32a1 expressions was observed in the (c1) brainstem. Illustrations of the Bregma sections in A–C are adapted from https:// mouse. brain- map. org/ exper iment/ thumb nails/ 10004 8576? image_ type= atlas. The red dashed squares in A–C indicate approximately the area displayed in a1–c1. The dashed squares were consistently placed on the right side of the schematic image in order to maximize the readability of the abbreviations, regardless of the position of the representative images. White arrows denote examples of co-expression. Scale bars: 200 µm, enlargements 100 µm. Abbreviated areas in a1–c1: Aco = anterior cortical amygdaloid nucleus, AHP = anterior hypothalamic area, posterior part, BLA = basolateral amygdaloid nucleus, anterior part, BMA = basomedial amygdaloid nucleus, anterior part, DG = dentate gyrus, DM = dorsomedial hypothalamic nucleus, Gi = gigantocellular reticular nucleus, IRt = intermediate reticular nucleus, ME = medial amygdaloid nucleus, MVeMC = medial vestibular nucleus, magnocellular part, MVePC = medial vestibular nucleus, parvicellular part, PLCo = posterolateral cortical amygdaloid nucleus, Pr = prepositus nucleus, Rt = reticular thalamic nucleus, S1J = primary somatosensory cortex, jaw region, S1ULP = primary somatosensory cortex, upper lip region, S2 = secondary somatosensory cortex, Sol = solitary tract, VL = ventrolateral thalamic nucleus, VMH = ventromedial hypothalamic nucleus, VPL = ventral posterolateral thalamic nucleus, VPM = ventral posteromedial thalamic nucleus, 3V = 3rd ventricle, 4V = 4th ventricle. For all other abbreviations, please see https:// mouse. brain- map. org/ exper iment/ thumb nails/ 10004 8576? image_ type= atlas C eder et al. BMC Neuroscience (2023) 24:32 Page 9 of 17 Fig. 4 (See legend on previous page.) Ceder et al. BMC Neuroscience (2023) 24:32 Page 10 of 17 substructures. Herein, in the bulk qRT-PCR, the cortex data was obtained using the 10X Genomics method. The includes the main olfactory bulb, accessory olfactory dataset was analyzed using SCANPY 1.9.1 [36] in Python bulb, anterior olfactory nucleus, orbital cortex and the 3.8.8 in similarity as described before [37] and the full frontal association cortex. Direct comparisons to the code can be found at https:// github. com/ Hanna hMWem dataset of Zeisel et al. [22] are therefore limited. More- an/ g lra3- e x pr e ssion- analy si s - in- t he- ner vo u s - s y st em. over, in contrast to the males’ striatum samples, the Firstly, all annotated neurons were isolated from the data- dissected female striatum only contained caudate puta- set to be used for basic preprocessing, resulting in 74,539 men and not nucleus accumbens, which could likely neurons and 27,998 genes. For gene filtering, all genes explain the differences in expression level in the striatum that were expressed in less than 3 cells (sc.pp.filter_genes) between females and males. Finally, amygdala, thalamus and all cells expressing less than 200 genes (sc.pp.filter_ and pituitary gland were dissected from animals with the cells) were excluded, resulting in 74,529 neurons and same housing and background, whereas the other qRT- 21,194 genes. Subsequently, for basic preprocessing, the PCR analyzed specimens were dissected from two dif- metrics of the general gene expression and mitochondrial ferent cohorts [34, 35]. However, the same expression genes were calculated (SCANPY, pp.calculate_gc_met- pattern between females and males was revealed inde- rics) [38]. By visualizing the distribution of the calculated pendent of this. metrics (SCANPY, pl.violin; Seaborn, jointplot), the cells with lower mitochondrial gene expression (SCANPY, Conclusions ‘pct_counts_mt’ < 20), high total counts (SCANPY, We herein conclude that Glra3 can be found in the cor- ‘log1p_total_counts’ > 6.5), and distributed gene counts tex, amygdala, striatum, hypothalamus, hippocampus, and broad gene capture (SCANPY, ‘logp_n_genes_by_ brainstem and the spinal cord in female and male mice. counts’ > 6.0, ‘pct_counts_in_top_50_genes’ < 50) were The expression pattern was verified using three differ - isolated. The dataset did not contain External RNA Con - ent mRNA-based methods. Furthermore, our analysis trols Consortium (ERCC) genes since the 10 × Genom- revealed that male mice display higher levels of Glra3 ics method does not include ERCC sequences, thus cells in the cortex, amygdala, hypothalamus, thalamus, hip- were not filtered based on expression criteria of these pocampus, cerebellum and the brainstem than females. sequences. All the inclusion criteria resulted in 72,020 Based on the expression patterns, future analyses may neurons and 21,194 genes to be used for the scRNA-seq investigate the functional role of GLRA3 in regulating analysis. Finally, the counts per cell was normalized to somatosensory modalities, such as pruriception, and fur- the medium number of counts (SCANPY, pp.normalize_ ther address the role of the subunit in nociception, both per_cell) followed by normalization (SCANPY, pp.log1p). in the brain and in the spinal cord. Single‑cell RNA sequencing analysis of Glra3‑expressing Methods cells Preprocessing of Zeisel et al. (2018) single‑cell RNA The expressions of Glra3, as well as excitatory Slc17a6 sequencing dataset (Vglut2) and inhibitory Slc32a1 (Viaat) markers, The expression of Glra3 in the nervous system was inves - were visualized in the respective nervous system area tigated in the Zeisel et al. (2018) scRNA-seq dataset [22]. (SCANPY, pl.DotPlot). Moreover, the prevalence The dataset ‘l5_all.loom’ was acquired from http:// linna of Glra3 expression (Glra3 considered expressed if rsson lab. org/ and contains expression data of 27,998 log1p > 0.1) was calculated for the respective nervous sys- genes in 160,796 single-cells from Vgat-Cre; tdTomato tem area. The expression patterns of Slc17a6 and Slc32a1 mice (with CD-1 and C57BL/6J background) to tar- were more extensively examined in all of the Glra3 neu- get inhibitory neurons and Wnt1-Cre; R26Tomato mice rons (a total of 628 neurons expressed Glra3) in the dif- (with C57BL/6J background) to isolate neurons in the ferent nervous system areas by visualization (SCANPY, peripheral and enteric nervous systems. The scRNA-seq pl.DotPlot) and occurrence calculations. (See figure on next page.) Fig. 5 Spatial expression analysis of Glra3 in the female spinal cord. The spatial Glra3 mRNA expression was examined with RNAscope using probes for Glra3 (teal), Slc17a6 (red) and Slc32a1 (red). Glra3 was expressed in the (A)cervical (C7), (B) thoracic ( T11), (C) lumbar (L5) and (D) sacral (S2) divisions of the spinal cord (A–D). Overlap with Slc17a6 and Slc32a1expressions could be observed in all divisions (a1, a2, b1, b2, c1, c2, d1, d2). Expression was found in the dorsal and ventral horns in all divisions. Glra3 could be detected in the ventral horn in the cervical, lumbar and sacral divisions. Illustrations of the spinal cord divisions in A–D are modified from https:// mouse. brain- map. org/ exper iment/ siv? id= 10005 0402& image Id= 10100 6525& image Type= atlas. The red dashed squares in A–D indicate approximately the enlarged images in a1-d2 (labelled with white text). No overview images are shown for the enlarged images labeled with red text. A–D Scale bars: 500 µm, a1–d2: scale bars 100 µm. White arrows denote examples of co-expression. Grey line in a1-d2 indicates boarder for lamina II and lamina II . 5Sp = lamina 5, 7Sp = lamina 7, 8Sp = lamina 8. For outer inner all other abbreviations, please see https:// mouse. brain- map. org/ exper iment/ siv? id= 10005 0402& image Id= 10100 6525& image Type= atlas C eder et al. BMC Neuroscience (2023) 24:32 Page 11 of 17 Fig. 5 (See legend on previous page.) Ceder et al. BMC Neuroscience (2023) 24:32 Page 12 of 17 Animals thalamus, hippocampus and amygdala were manually Protocols related to animal use in this study were dissected with guidance from a mouse brain atlas [40] approved by the local animal research ethical commit- and collected. The whole cerebellum and brainstem were tee (Uppsala djurförsöksetiska nämnd) and followed the collected. Moreover, the following tissues were collected: Swedish Animal Welfare Act [Svensk författningssamling spinal cord (late thoracic to sacral divisions), heart, small (SFS) 2018:1192], The Swedish Animal Welfare Ordi - intestine, kidney, liver, lung, spleen, testes, thymus, and nance (SFS 2019:66) and the Regulations and General uterus. For the additional five male mice dissected for Advice for Laboratory Animals (SJVFS 2019:9, Saknr this paper, only amygdala, pituitary gland, and thalamus L 150). Both female and male C57BL/6J mice (Taconic, were dissected, as described for the females, to comple- Denmark) were included in the analysis. The mice were ment the other male panel [34, 35]. housed with littermates in approximately 501 cm cages All tissues were collected within 10–15 min after sacri- (maximum 5 mice in per cage), in room temperature fice and stored in RNAprotect© Tissue Reagent (Qiagen, ranging between 20 and 24 °C and humidity of 45–65% Germany) for 2 h at room temperature. All samples were on a 12-h light:dark cycle with lights on at 6 am. All ani- then frozen at -80 °C before further processing. mals were provided food (Diet Pellets, Scanbur, Sweden) and tap water ad libitum. All procedures were planned RNA extraction and cDNA synthesis and executed to minimize stress, and euthanasia was per- Total RNA was extracted using Absolutely RNA Mini formed during the light period of the light:dark cycle. kit (Qiagen, Germany) according to the manufacturer’s protocol. RNA concentrations were measured using Tissue dissection for qRT‑PCR ND-1000 spectrophotometer (NanoDrop Technologies, Tissues from five adult male C57BL/6J mice (10– USA). The cDNA synthesis was performed using the 14 weeks) had previously been collected and prepared Applied Biosystems High Capacity RNA-to-cDNA kit as specified in [34, 35], where the striatum samples (Invitrogen, USA) following manufacturer’s instructions. comprised of the caudate putamen and nucleus accum- 2 μg RNA template was used for the reaction and the bens. The gDNA was previous collected and was a gift cDNA samples were diluted to 10 ng/μl. from Prof. Robert Fredriksson [39]. To add to this tissue mRNA panel, five adult female (14 weeks old) and five adult male (10–11 weeks old) C57BL/6J mice were eutha- Primer design and quantitative real‑time PCR (qRT‑PCR) nized via cervical dislocation, without prior treatment, Primers were designed using Primer3 (Glra3) [41] or during the light period. All tissues were collected on ice. Beacon Design 8 (Premier Biosoft) (reference house- The following tissues/areas were collected from the five keeping genes). The primers were screened using BLAST females. The whole brain was scooped out leaving the and global alignments [42] to avoid primer pairs that can majority of the main olfactory bulb in the scull. The pitui - cause non-specific amplification. Glra3 primers: for - tary gland was collected from sella turcica and the hypo- ward 5′-cggaagcttttgcactggag-3′, reverse 5′-tggaaccacac- thalamus was collected from the brain using forceps. The catccttgg-3′. Reference housekeeping genes: ribosomal brain was then placed in a mouse brain matrix (Activa- protein L19 (Rpl19) forward 5′-aatcgccaatgccaactc-3′, tional Systems Inc., Warren, MI, USA; 1 mm) and sliced reverse 5′-ggaatggacagtcacagg-3′, Peptidylprolyl isomeras manually using matrices blades (ALTO Matrix Cutting A (Cyclo) forward 5′-tttgggaaggtgaaagaagg-3′, reverse Blades, AgnTho’s, Lidingö, Sweden). From coronal sec - 5′-acagaaggaatggtttgatgg-3′, glyceraldehyde-3-phosphate tions, the most frontal part of the cortex (herein cortex; dehydrogenase (Gapdh) forward 5′-gccttccgtgttcctacc-3′, containing main olfactory bulb, accessory olfactory bulb, reverse 5′-gcctgcttcaccaccttc-3′ and actin-related protein anterior olfactory nucleus, orbital cortex, and frontal 1B (Actb) forward 5′-ccttcttgggtatggaatcctgtg-3′, reverse association cortex), caudate putamen (herein striatum), 5′-cagcactgtgttggcatagagg-3′. (See figure on next page.) Fig. 6 Spatial expression analysis of Glra3 in the male spinal cord. The spatial Glra3 mRNA expression was examined with RNAscope using probes for Glra3 (teal), Slc17a6 (red) and Slc32a1 (red). Glra3 was expressed in the (A) cervical (C7), (B) thoracic ( T11), (C) lumbar (L5) and (D) sacral (S2) divisions of the spinal cord (A–D). Overlap with Slc17a6 and Slc32a1 expressions could be observed in all divisions (a1, a2, b1, b2, c1, c2, d1, d2). Glra3 expression was found in the dorsal and ventral horns in all divisions. Glra3 could be detected in the ventral horn in the cervical, lumbar and sacral divisions. Illustrations of the spinal cord divisions in A–D are modified from https:// mouse. brain- map. org/ exper iment/ siv? id= 10005 0402& image Id= 10100 6525& image Type= atlas. The red dashed squares in A–D indicate approximately the area displayed in a1–d2 (labelled with white text). No overview images are shown for the enlarged images labeled with red text. A–D Scale bars: 500 µm, a1–d2: scale bars 100 µm. White arrows denote examples of co-expression. Grey line in a1-d2 indicates boarder for lamina II and lamina II . 5Sp = lamina 5, 6Sp = lamina 6, outer inner 7Sp = lamina 7, 8Sp = lamina 8. For all other abbreviations, please see https:// mouse. brain- map. org/ exper iment/ siv? id= 10005 0402& image Id= 10100 6525& image Type= atlas C eder et al. BMC Neuroscience (2023) 24:32 Page 13 of 17 Fig. 6 (See legend on previous page.) Ceder et al. BMC Neuroscience (2023) 24:32 Page 14 of 17 Table 2 Summary of Glra3 expression in the central and The log2 fold difference to the genomic DNA expression peripheral nervous system of Glra3 was calculated for all tissues and presented in £ the combined scatter-bar-plot graph (mean log2 differ - Areas scRNA‑seq qRT‑PCR RNAscope ence against gDNA expression of Glra3 ± SEM). Cortex + + + Amygdala + + + In situ hybridization tissue preparation Striatum + + + (males) Two adult female (13 weeks old) and two adult male Pallidum + NA + (11–13 weeks old) C57BL/6J mice were intraperitoneally Hypothalamus + + + injected with 0.6 ml (1:1) Ketamin (Ketalar, 10 mg/ml, Pituitary gland NA + NA Pfizer, Sweden) and Medetomidine (Domitor, 1 mg/ml, Hippocampus + + + Orion Pharma, Sweden) and subsequently perfused with Brainstem + + + autoclaved ice-cold 1 × PBS. To minimize the risk of con- Cerebellum ND + ND tamination and altered gene expression, the following Spinal cord + + + steps were performed as quickly as possible in autoclaved Dorsal root ganglia ND NA NA ice-cold 1 × PBS; the whole brains and all divisions of the + expression, NA not analyzed, ND not detected. Zeisel et al. (2018) dataset [22] spinal cord were dissected and cleaned from meninges, followed by embedding in optimal cutting temperature (OCT) medium (Bio-Optica, Italy) and snap-frozen on Glra3 expression was determined using qRT-PCR. dry ice in -80 °C isopentane (Sigma-Aldrich, Germany). Final volume for each reaction was 20 μl containing The tissues were stored in -80 °C until sectioning. The 3.6 μl 10 × DreamTaq Buffer (Thermo Fisher Scientific, brains were cryo-sectioned (Leica Cryocut 1800, Leica, USA), 0.2 μl of 20 mM dNTP mix (Invitrogen, USA), 1 μl Germany) into 18 µm and the spinal cords into 14 µm DMSO, 0.5 μl SYBR Green (1:10,000, Invitrogen, USA) in sections and collected onto Superfrost Plus (Thermo 1 × TE buffer (pH 7.8), 0.08 μl DreamTaq polymerase (5 Scientific, USA) slides. To prevent mRNA degradation U/μl, Thermo Fisher Scientific, USA), 0.05 μl of forward and contamination, the completed series were stored at and reverse primer (100 pmol/μl) and 5 μl cDNA (10 ng/ -21 °C until sectioning was completed. The slides were μl). The volume was adjusted with sterile water. An iCy - thereafter stored at -80 °C until the RNAscope Fluores- cler real-time detection instrument (Bio-Rad, USA) was cent Multiplex kit (Advanced Cell Diagnostics (ACD), used with the following settings: initial denaturation USA, cat # 320850) protocol commenced. for 30 s at 95 °C, 45 cycles of 10 s at 95 °C, 30 s at 55 °C for housekeeping genes or 55.7 °C for Glra3 and 30 s at 72 °C. A melting curve was generated by heating from 55 Fluorescent in situ hybridization to 95 °C with 0.5 °C increments at 10 s dwell time and Fluorescent in situ hybridization was performed to tar- a plate read at each temperature. All qRT-PCR were run get the expression of Glra3 in various tissues using the in triplicates and a negative control and genomic DNA RNAscope Fluorescent Multiplex kit (cat#: 320850, ACD, (10 ng/ul) were included on each plate. Cycle threshold USA) in accordance with ACD guidelines for fresh frozen (Ct) values were collected via the CFX Maestro (Bio-Rad, tissues with minor modifications [24] and as described USA) and primer efficiencies were calculated via Lin - previously [37]. In brief: the slides to be used were taken RegPCR software. The melting curves were compared from -80 °C and immediately fixated in room temperature with the negative control to verify that only one product 4% PFA in 1 × PBS (Histolab, Sweden) for 15 min before was amplified. The delta Ct method for multiple refer - being washed in autoclaved 1 × PBS for 2 min. The tis - ence genes (according to [23]) was used to calculate the sues were thereafter dehydrated in a step-wise increase of normalized and relative mRNA expression of Glra3, EtOH concentration; 3 min in 50%, 3 min in 70% and two and differences in primer efficiency were accounted for. times for 5 min in 100% (Merck KGaA, Damstadt, Ger- Biological outliers in nervous system tissues (one for many). The slides were placed at room temperature for hippocampus and one for cerebellum from male mice) 5 min to dry whereafter a hydrophobic barrier was made were removed using the Grubbs outlier test with α = 0.05 around the slide area of interest (2 females and 2 males; before proceeding and 45 cycles were set as cut-off. The brain: 2 sections/brain area of interest (Bregma 0.98, -1.34 same settings for the Grubbs outlier test and cycle thresh- and -6.84 mm [20] https:// mouse. brain- map. org/ exper old cut-off were used for the identification of biological iment/ thumb nails/ 10004 8576? image_ type= atlas) from outliers in the visceral organs. The following biological each animal; spinal cord: 4 sections/spinal cord division outliers were consequently removed; heart (two females (cervical, thoracic, lumbar and sacral) from each animal [9] and two males), lung (two males), liver (one female and http s :// mou s e. brain- map. org/ exp er iment/ siv? id= 10005 one male), spleen (one female) and kidney (one female). 0402& image Id= 10100 6525& image Type= atlas) using an C eder et al. BMC Neuroscience (2023) 24:32 Page 15 of 17 Abbreviations ImmeEdge pen (Vector Laboratories, USA). The sections DRG Dorsal root ganglia were thereafter incubated in Protease IV for 40 min at Glra3 Glycine receptor alpha3 subunit room temperature, followed by washing three times for GlyR Glycine receptor 5 min in autoclaved 1 × PBS. The treatment was followed GLY T2 Glycine transporter subtype 2 HPA Hypothalamic–pituitary–adrenal by incubation with the target probes; Glra3: 490591- qRT-PCR Quantitative real-time PCR C2 and Slc17a6 (Vglut2): 319171-C3 or Slc32a1 (Viaat): RFU Relevant fluorescence unit 319191-C3 (1:50 in probe diluent, cat#: 300041) for 2 h scRNA-seq Single -cell RNA sequencing Vglut2 V esicular glutamate transporter 2 (Slc17a6) at 40 °C in a hybridization oven (HybEZ II Oven, ACD, Viaat Vesicular inhibitory amino acid transporter (Slc32a1) USA) (brain: 1 section/area from each animal per assay (Bregma 0.98, -1.34 and -6.84 mm [20] https:// mouse. Supplementary Information brain- ma p. or g/ e x p er imen t/ t h umb nail s/ 10004 8576? The online version contains supplementary material available at https:// doi. image_ type= atlas); spinal cord: 2 sections/division (cervi- org/ 10. 1186/ s12868- 023- 00800-9. cal, thoracic, lumbar and sacral [9] https:// mouse. brain- map. org/ exper iment/ siv? id= 10005 0402& image Id= 10100 Additional file 1. Additional figures, Fig. S1‑S2. Additional Table, Table S1. 6525& image Type= atlas) from each animal per assay). The following amplification steps were performed at 40 °C in an oven and the sections were washed two times for 2 min Acknowledgements We acknowledge Robert Fredriksson for shared material and Elena Witzemann in room temperature washing buffer between each ampli - for proofreading. fication step; AMP 1-FL for 30 min, AMP 2-FL for 15 min, AMP 3-FL for 30 min and AMP 4-FL for 15 min. Lastly, Author contributions the slides were washed two times for 2 min in washing Conceptualization: MCL, MMC; Formal Analysis: MMC, HMW; Funding Acquisi- tion: MCL; Investigation: MMC, HMW, EJ, KH, KAM, ER; Methodology: MMC, buffer before 30 s incubation in DAPI and mounting in HMW, MCL; Project Administration: MMC, MCL; Resources: MCL; Supervision: Anti-Fade Fluorescence Mounting Medium (Abcam, UK). MMC, MCL; Validation: MMC, HMW, MCL; Visualization: MMC, HMW; Writing— The slides were covered with glass slides (Menzel-Gläser, Original Draft Preparation: MMC, HMW, MCL; Writing—Review and Editing: All authors. All authors read and approved the final manuscript. Germany) and were left at 4 °C to dry. The slides were stored at this temperature until imaging. Funding Open access funding provided by Uppsala University. This work was sup- ported by the Swedish Brain Foundation, the Swedish Research Council (2016- In situ image acquisition 00851 and 2022-00960) and Uppsala University. Images of the RNAscope treated sections were acquired Availability of data and materials with wide field 20 × magnification using an Axio Imager. The scRNA-seq data was acquired from Zeisel et al. (2018) scRNA-seq dataset Z2 (Zeiss, Germany). Whole section images were [22], where the raw sequence data is deposited in the sequence read archive under accession SRP135960, available at https:// www. ncbi. nlm. nih. gov/ sra/ acquired as tiles in the DAPI (150 ms), Cy3 (Glra3 detec- SRP13 5960. The dataset ‘l5_all.loom’ was acquired from (http:// linna rsson lab. tion, 4000 ms) and Cy5 (Slc17a6 or Slc32a1 detection, org/). The dataset was analyzed using SCANPY 1.9.1 [36] in Python 3.8.8 in 900 ms) channels. The images were handled for figure similarity as described before [37] and the full code can be found at https:// github. com/ Hanna hMWem an/ glra3- expre ssion- analy sis- in- the- nervo us- representation using the ZEISS ZEN 3.3 (blue edition) system. All qRT-PCR data generated or analyzed during this study are included software, where the area outliners of the targeted brain in this published article and its additional files. structures were determined using the Allen mouse brain atlas (https:// mouse. brain- map. org/ exper iment/ thumb Declarations nails/ 10004 8576? image_ type= atlas). Ethics approval and consent to participate Protocols related to animal use in this study were approved by the local animal research ethical committee (Uppsala djurförsöksetiska nämnd) and followed Statistics the Swedish Animal Welfare Act [Svensk författningssamling (SFS) 2018:1192], The obtained relative mRNA values for female and male the Swedish Animal Welfare Ordinance (SFS 2019:66) and the Regulations and mice were checked for normality using the Shapiro- General Advice for Laboratory Animals (SJVFS 2019:9, Saknr L 150; permit num- bers C419/12, C39/16, 5.2.18-17971/19, 5.8.18-19421/19 and 5.8.18-11551/19). Wilk test before proceeding with the appropriate analy- All methods are reported in accordance with ARRIVE guidelines Essential 10, sis. Kruskal-Wallis test and Mann-Whitney U-test were where relevant. Both female and male C57BL/6J mice ( Taconic, Denmark) were used to determine expressional differences against the included in the analysis. The mice were housed with littermates in approxi- mately 501 cm cages (maximum 5 mice in per cage). The mice were housed background and tissues/areas of the nervous system in a room temperature ranging between 20 and 24 °C and humidity of 45–65% of both females and males (Additional file 1: Table S1). on a 12-h light:dark cycle with lights on at 6 am. All animals were provided Furthermore, two-tailed Mann-Whitney U-test or food (diet pellets, Scanbur, Sweden) and tap water ad libitum. 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Journal
BMC Neuroscience – Springer Journals
Published: Jun 1, 2023
Keywords: Glycine; Glra3; Brain; Spinal cord; Mice; Sex-dependent differences