Spinal glycine receptor alpha 3 cells communicate sensations of chemical itch in hairy skin.

Glycinergic neurons regulate nociceptive and pruriceptive signaling in the spinal cord, and the identity and role of the glycine-regulated neurons are not fully known. Herein, we have characterized spinal glycine receptor alpha 3 (Glra3) subunit-expressing neurons in Glra3-Cre female and male mice. Glra3-Cre(+) neurons express Glra3, are located mainly in laminae III‒VI, and respond to glycine. Chemogenetic activation of spinal Glra3-Cre(+) neurons induced biting/licking, stomping, and guarding behaviors, indicative of both a nociceptive and pruriceptive role for this population. Chemogenetic inhibition did not affect mechanical or thermal responses, but reduced behaviors evoked by compound 48/80 and chloroquine, revealing a pruriceptive role for these neurons. Spinal cells activated by compound 48/80 or chloroquine express Glra3, further supporting the phenotype. Retrograde tracing revealed that spinal Glra3-Cre(+) neurons receive input from afferents associated with pain and itch, and dorsal root stimulation validated the monosynaptic input. In conclusion, these results show that spinal Glra3(+) neurons contribute to acute communication of compound 48/80- and chloroquine-induced itch in hairy skin.Significance Statement Spinal glycinergic neurons regulate itch (pruriception), suggesting that components of the glycinergic system have great potential as drug targets to treat pruritus. Nonetheless, thus far, the pruriceptive roles of any of the glycine receptor (GLR) subunits have not been evaluated. Here, we successfully linked the Glra3-Cre populations to a pro-pruriceptive role in itch, indicating that GLRA3-expressing neurons may be a potential novel target for itch treatment. The spontaneous stomping and guarding behaviors observed from activating the Glra3-Cre populations are indicative of a role in sensory hypersensitivity and hence, raises questions regarding the hypersensitivity involvement of these populations for future investigations.

The glycine receptor alpha 3 subunit mRNA expression shows sex-dependent differences in the adult mouse brain.

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 composed 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, amygdala, striatum, hypothalamus, thalamus, pituitary gland, hippocampus, cerebellum, brainstem, and spinal cord. Additionally, 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 suggested from the single-cell data. CONCLUSIONS: Glra3 expression is low but widespread in the mouse central nervous system. Clear sex-dependent differences 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.

Adult spinal Dmrt3 neurons receive direct somatosensory inputs from ipsi- and contralateral primary afferents and from brainstem motor nuclei.

In the spinal cord, sensory-motor circuits controlling motor activity are situated in the dorso-ventral interface. The neurons identified by the expression of the transcription factor Doublesex and mab-3 related transcription factor 3 (Dmrt3) have previously been associated with the coordination of locomotion in horses (Equus caballus, Linnaeus, 1758), mice (Mus musculus, Linnaeus, 1758), and zebrafish (Danio rerio, F. Hamilton, 1822). Based on earlier studies, we hypothesized that, in mice, these neurons may be positioned to receive sensory and central inputs to relay processed commands to motor neurons. Thus, we investigated the presynaptic inputs to spinal Dmrt3 neurons using monosynaptic retrograde replication-deficient rabies tracing. The analysis showed that lumbar Dmrt3 neurons receive inputs from intrasegmental neurons, and intersegmental neurons from the cervical, thoracic, and sacral segments. Some of these neurons belong to the excitatory V2a interneurons and to plausible Renshaw cells, defined by the expression of Chx10 and calbindin, respectively. We also found that proprioceptive primary sensory neurons of type Ia2, Ia3, and Ib, defined by the expression of calbindin, calretinin, and Brn3c, respectively, provide presynaptic inputs to spinal Dmrt3 neurons. In addition, we demonstrated that Dmrt3 neurons receive inputs from brain areas involved in motor regulation, including the red nucleus, primary sensory-motor cortex, and pontine nuclei. In conclusion, adult spinal Dmrt3 neurons receive inputs from motor-related brain areas as well as proprioceptive primary sensory neurons and have been shown to connect directly to motor neurons. Dmrt3 neurons are thus positioned to provide sensory-motor control and their connectivity is suggestive of the classical reflex pathways present in the spinal cord.

A phylogenetic analysis between humans and D. melanogaster: A repertoire of solute carriers in humans and flies.

The solute carrier (SLC) superfamily is the largest group of transporters in humans, with the role to transport solutes across plasma membranes. The SLCs are currently divided into 65 families with 430 members. Here, we performed a detailed mining of the SLC superfamily and the recent annotated family of "atypical" SLCs in human and D. melanogaster using Hidden Markov Models and PSI-BLAST. Our analyses identified 381 protein sequences in D. melanogaster and of those, 55 proteins have not been previously identified in flies. In total, 11 of the 65 human SLC families were found to not be conserved in flies, while a few families are highly conserved, which perhaps reflects the families' functions and roles in cellular pathways. This study provides the first collection of all SLC sequences in D. melanogaster and can serve as a SLC database to be used for classification of SLCs in other phyla.

The Fly Homologue of MFSD11 Is Possibly Linked to Nutrient Homeostasis and Has a Potential Role in Locomotion: A First Characterization of the Atypical Solute Carrier CG18549 in Drosophila Melanogaster.

Cellular transport and function are dependent on substrate influx and efflux of various compounds. In humans, the largest superfamily of transporters is the SoLute Carriers (SLCs). Many transporters are orphans and little to nothing is known about their expression and/or function, yet they have been assigned to a cluster called atypical SLCs. One of these atypical SLCs is MFSD11. Here we present a first in-depth characterization of the MFSD11, CG18549. By gene expression and behavior analysis on ubiquitous and brain-specific knockdown flies. CG18549 knockdown flies were found to have altered adipokinetic hormone and adipokinteic hormone receptor expression as well as reduced vesicular monoamine transporter expression; to exhibit an altered locomotor behavior, and to have an altered reaction to stress stimuli. Furthermore, the gene expression of CG18549 in the brain was visualized and abundant expression in both the larvae and adult brain was observed, a result that is coherent with the FlyAtlas Anatomy microarray. The exact mechanism behind the observed behaviors is not fully understood, but this study provides new insights into the expression and function of CG18549. Clearly, these results provide a strong example as to why it is vital to fully characterize orphan transporters and through that gain knowledge about the body during normal condition and disease.

Differentiation of two human neuroblastoma cell lines alters SV2 expression patterns.

BACKGROUND: The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation. METHODS: Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed. RESULTS: The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells. CONCLUSION: The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function.

CG4928 Is Vital for Renal Function in Fruit Flies and Membrane Potential in Cells: A First In-Depth Characterization of the Putative Solute Carrier UNC93A.

The number of transporter proteins that are not fully characterized is immense. Here, we used Drosophila melanogaster and human cell lines to perform a first in-depth characterization of CG4928, an ortholog to the human UNC93A, of which little is known. Solute carriers regulate and maintain biochemical pathways important for the body, and malfunctioning transport is associated with multiple diseases. Based on phylogenetic analysis, CG4928 is closely related to human UNC93A and has a secondary and a tertiary protein structure and folding similar to major facilitator superfamily transporters. Ubiquitous knockdown of CG4928 causes flies to have a reduced secretion rate from the Malpighian tubules; altering potassium content in the body and in the Malpighian tubules, homologous to the renal system; and results in the development of edema. The edema could be rescued by using amiloride, a common diuretic, and by maintaining the flies on ion-free diets. CG4928-overexpressing cells did not facilitate the transport of sugars and amino acids; however, proximity ligation assay revealed that CG4928 co-localized with TASK1 channels. Overexpression of CG4928 resulted in induced apoptosis and cytotoxicity, which could be restored when cells were kept in high-sodium media. Furthermore, the basal membrane potential was observed to be disrupted. Taken together, the results indicate that CG4928 is of importance for generating the cellular membrane potential by an unknown manner. However, we speculate that it most likely acts as a regulator or transporter of potassium flows over the membrane.

Glucose Availability Alters Gene and Protein Expression of Several Newly Classified and Putative Solute Carriers in Mice Cortex Cell Culture and D. melanogaster.

Many newly identified solute carriers (SLCs) and putative transporters have the possibility to be intricately involved in glucose metabolism. Here we show that many transporters of this type display a high degree of regulation at both mRNA and protein level following no or low glucose availability in mouse cortex cultures. We show that this is also the case in Drosophila melanogaster subjected to starvation or diets with different sugar content. Interestingly, re-introduction of glucose to media, or refeeding flies, normalized the gene expression of a number of the targets, indicating a fast and highly dynamic control. Our findings demonstrate high conservation of these transporters and how dependent both cell cultures and organisms are on gene and protein regulation during metabolic fluctuations. Several transporter genes were regulated simultaneously maybe to initiate alternative metabolic pathways as a response to low glucose levels, both in the cell cultures and in D. melanogaster. Our results display that newly identified SLCs of Major Facilitator Superfamily type, as well as the putative transporters included in our study, are regulated by glucose availability and could be involved in several cellular aspects dependent of glucose and/or its metabolites. Recently, a correlation between dysregulation of glucose in the central nervous system and numerous diseases such as obesity, type 2 diabetes mellitus as well as neurological disease such as Alzheimer's and Parkinson's diseases indicate a complex regulation and fine tuning of glucose levels in the brain. The fact that almost one third of transporters and transporter-related proteins remain orphans with unknown or contradictive substrate profile, location and function, pinpoint the need for further research about them to fully understand their mechanistic role and their impact on cellular metabolism.

Nutritional Stress Induced by Amino Acid Starvation Results in Changes for Slc38 Transporters in Immortalized Hypothalamic Neuronal Cells and Primary Cortex Cells.

Amino acid sensing and signaling is vital for cells, and both gene expression and protein levels of amino acid transporters are regulated in response to amino acid availability. Here, the aim was to study the regulation of all members of the SLC38 amino acid transporter family, Slc38a1-11, in mouse brain cells following amino acid starvation. We reanalyzed microarray data for the immortalized hypothalamic cell line N25/2 subjected to complete amino acid starvation for 1, 2, 3, 5, or 16 h, focusing specifically on the SLC38 family. All 11 Slc38 genes were expressed in the cell line, and Slc38a1, Slc38a2, and Slc38a7 were significantly upregulated at 5 h and most strongly at 16 h. Here, protein level changes were measured for SLC38A7 and the orphan family member SLC38A11 which has not been studied under different amino acid starvation condition at protein level. At 5 h, no significant alteration on protein level for either SLC38A7 or SLC38A11 could be detected. In addition, primary embryonic cortex cells were deprived of nine amino acids, the most common amino acids transported by the SLC38 family members, for 3 h, 7 h or 12 h, and the gene expression was measured using qPCR. Slc38a1, Slc38a2, Slc38a5, Slc38a6, Slc38a9, and Slc38a10 were upregulated, while Slc38a3 and Slc38a7 were downregulated. Slc38a8 was upregulated at 5 h and downregulated at 12 h. In conclusion, several members from the SLC38 family are regulated depending on amino acid levels and are likely to be involved in amino acid sensing and signaling in brain.

The Neuronal and Peripheral Expressed Membrane-Bound UNC93A Respond to Nutrient Availability in Mice.

Many transporters such as the solute carriers belonging to the Major facilitator superfamily Pfam clan are orphans in that their tissue and cellular localization as well as substrate profile and function are still unknown. Here we have characterized the putative solute carrier UNC93A. We aimed to investigate the expression profile on both protein and mRNA level of UNC93A in mouse since it has not been clarified. UNC93A staining was found in cortex, hippocampus and cerebellum. It was found to be expressed in many neurons, but not all, with staining located in close proximity to the plasma membrane. Furthermore, we aimed to extend the starvation data available for Unc93a in hypothalamic cell cultures from mouse. We investigated the Unc93a alterations with focus on amino acid deprivation in embryonic cortex cells from mice as well as 24 h starvation in adult male mice and compared it to recently studied putative and known solute carriers. Unc93a expression was found both in the brain and peripheral organs, in low to moderate levels in the adult mice and was affected by amino acid deprivation in embryonic cortex cultures and starvation in in vivo samples. In conclusion, the membrane-bound UNC93A is expressed in both the brain and peripheral tissues and responds to nutrient availability in mice.