Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.

Pitfalls of using sequence databases for heterologous expression studies - a technical review.

Synthesis of DNA fragments based on gene sequences that are available in public resources has become an efficient and affordable method that has gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterisation of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: (1) Not every gene has yet been annotated; (2) not all gene annotations are necessarily correct; (3) transcripts may contain automated corrections; (4) there are mismatches between gene, mRNA and protein sequences; and (5) splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems.

Human airway tuft cells influence the mucociliary clearance through cholinergic signalling.

BACKGROUND: Airway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive. METHODS: We performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed. RESULTS: Tuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance. CONCLUSIONS: We were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.

Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, ß, γ, δ), forming heterotrimeric αßγ- or δßγ-ENaCs. Although the physiology of αßγ-ENaC is well understood, for decades the field has stalled with respect to δßγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δßγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αßγ- and δßγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

Evolution of the Autism-Associated Neuroligin-4 Gene Reveals Broad Erosion of Pseudoautosomal Regions in Rodents.

Variants in genes encoding synaptic adhesion proteins of the neuroligin family, most notably neuroligin-4, are a significant cause of autism spectrum disorders in humans. Although human neuroligin-4 is encoded by two genes, NLGN4X and NLGN4Y, that are localized on the X-specific and male-specific regions of the two sex chromosomes, the chromosomal localization and full genomic sequence of the mouse Nlgn4 gene remain elusive. Here, we analyzed the neuroligin-4 genes of numerous rodent species by direct sequencing and bioinformatics, generated complete drafts of multiple rodent neuroligin-4 genes, and examined their evolution. Surprisingly, we find that the murine Nlgn4 gene is localized to the pseudoautosomal region (PAR) of the sex chromosomes, different from its human orthologs. We show that the sequence differences between various neuroligin-4 proteins are restricted to hotspots in which rodent neuroligin-4 proteins contain short repetitive sequence insertions compared with neuroligin-4 proteins from other species, whereas all other protein sequences are highly conserved. Evolutionarily, these sequence insertions initiate in the clade eumuroidea of the infraorder myomorpha and are additionally associated with dramatic changes in noncoding sequences and gene size. Importantly, these changes are not exclusively restricted to neuroligin-4 genes but reflect major evolutionary changes that substantially altered or even deleted genes from the PARs of both sex chromosomes. Our results show that despite the fact that the PAR in rodents and the neuroligin-4 genes within the rodent PAR underwent massive evolutionary changes, neuroligin-4 proteins maintained a highly conserved core structure, consistent with a substantial evolutionary pressure preserving its physiological function.

An extracellular acidic cleft confers profound H+-sensitivity to epithelial sodium channels containing the δ-subunit in Xenopus laevis.

The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from αßγ- or δßγ-subunit combinations, we demonstrate that Xenopus δßγ-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus αßγ-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the δ-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus δßγ-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release.

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon-specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca(2+)-buffer EGTA, suggesting that synaptic ribbons mediate nano-domain coupling of Ca(2+) channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano-domains that position release-ready synaptic vesicles adjacent to Ca(2+) channels.

Presynaptic Neuronal Pentraxin Receptor Organizes Excitatory and Inhibitory Synapses.

Three neuronal pentraxins are expressed in brain, the membrane-bound "neuronal pentraxin receptor" (NPR) and the secreted proteins NP1 and NARP (i.e., NP2). Neuronal pentraxins bind to AMPARs at excitatory synapses and play important, well-documented roles in the activity-dependent regulation of neural circuits via this binding activity. However, it is unknown whether neuronal pentraxins perform roles in synapses beyond modulating postsynaptic AMPAR-dependent plasticity, and whether they may even act in inhibitory synapses. Here, we show that NPR expressed in non-neuronal cells potently induces formation of both excitatory and inhibitory postsynaptic specializations in cocultured hippocampal neurons. Knockdown of NPR in hippocampal neurons, conversely, dramatically decreased assembly and function of both excitatory and inhibitory postsynaptic specializations. Overexpression of NPR rescued the NPR knockdown phenotype but did not in itself change synapse numbers or properties. However, the NPR knockdown decreased the levels of NARP, whereas NPR overexpression produced a dramatic increase in the levels of NP1 and NARP, suggesting that NPR recruits and stabilizes NP1 and NARP on the presynaptic plasma membrane. Mechanistically, NPR acted in excitatory synapse assembly by binding to the N-terminal domain of AMPARs; antagonists of AMPA and GABA receptors selectively inhibited NPR-induced heterologous excitatory and inhibitory synapse assembly, respectively, but did not affect neurexin-1ß-induced synapse assembly as a control. Our data suggest that neuronal pentraxins act as signaling complexes that function as general trans-synaptic organizers of both excitatory and inhibitory synapses by a mechanism that depends, at least in part, on the activity of the neurotransmitter receptors at these synapses. SIGNIFICANCE STATEMENT: Neuronal pentraxins comprise three neuronal proteins, neuronal pentraxin receptor (NPR) which is a type-II transmembrane protein on the neuronal surface, and secreted neuronal pentraxin-1 and NARP. The general functions of neuronal pentraxins at synapses have not been explored, except for their basic AMPAR binding properties. Here, we examined the functional role of NPR at synapses because it is the only neuronal pentraxin that is anchored to the neuronal cell-surface membrane. We find that NPR is a potent inducer of both excitatory and inhibitory heterologous synapses, and that knockdown of NPR in cultured neurons decreases the density of both excitatory and inhibitory synapses. Our data suggest that NPR performs a general, previously unrecognized function as a universal organizer of synapses.

Latrophilins function as heterophilic cell-adhesion molecules by binding to teneurins: regulation by alternative splicing.

Latrophilin-1, -2, and -3 are adhesion-type G protein-coupled receptors that are auxiliary α-latrotoxin receptors, suggesting that they may have a synaptic function. Using pulldowns, we here identify teneurins, type II transmembrane proteins that are also candidate synaptic cell-adhesion molecules, as interactors for the lectin-like domain of latrophilins. We show that teneurin binds to latrophilins with nanomolar affinity and that this binding mediates cell adhesion, consistent with a role of teneurin binding to latrophilins in trans-synaptic interactions. All latrophilins are subject to alternative splicing at an N-terminal site; in latrophilin-1, this alternative splicing modulates teneurin binding but has no effect on binding of latrophilin-1 to another ligand, FLRT3. Addition to cultured neurons of soluble teneurin-binding fragments of latrophilin-1 decreased synapse density, suggesting that latrophilin binding to teneurin may directly or indirectly influence synapse formation and/or maintenance. These observations are potentially intriguing in view of the proposed role for Drosophila teneurins in determining synapse specificity. However, teneurins in Drosophila were suggested to act as homophilic cell-adhesion molecules, whereas our findings suggest a heterophilic interaction mechanism. Thus, we tested whether mammalian teneurins also are homophilic cell-adhesion molecules, in addition to binding to latrophilins as heterophilic cell-adhesion molecules. Strikingly, we find that although teneurins bind to each other in solution, homophilic teneurin-teneurin binding is unable to support stable cell adhesion, different from heterophilic teneurin-latrophilin binding. Thus, mammalian teneurins act as heterophilic cell-adhesion molecules that may be involved in trans-neuronal interaction processes such as synapse formation or maintenance.

The molecular basis of the specificity and cross-reactivity of the NeuN epitope of the neuron-specific splicing regulator, Rbfox3.

Ever since its description and the generation of its defining antibody some 20 years ago, NeuN (Neural Nuclei) has been an invaluable tool for developmental neuroscientist sand neuropathologists to identify neurons and follow their normal or malignant development [corrected].The recent identification of the splicing factor Rbfox3 as the molecule constituting the genuine NeuN epitope has opened up a novel perspective on NeuN immunostaining and its interpretation. Here, we briefly review these recent developments, and we provide a series of data that allow to rationalize the specificity of the NeuN/A60 antibody on aldehyde-fixed tissues on the one hand, and its cross-reactivity with Synapsin I and R3hdm2 on Western blots on the other. We argue that rather than being considered as a mere marker for mature neurons, Rbfox3-mediated NeuN/A60 immunoreactivity may provide a window onto neuronal biology. Specifically, we hypothesize that the phosphorylation-dependent antigenicity of the Rbfox3/NeuN epitope should allow to visualize neuronal physiology realized through Rbfox3, including splicing, on the single-cell level.

Rare Superior and Middle Trunk Fusion Accompanied by Altered Division Rearrangement Results in a Unique Brachial Plexus Variant: A Case Report.

During routine dissections of cadavers as part of the medical curriculum, we identified a rare unilateral variation in the brachial plexus on the right side of a female body donor. This variation consisted of four unusual changes to the regular pattering of nerve bundles and the dorsal scapular artery permeating the complex neural network. The variation included contributions of root C4 to the plexus by a root C4/C5 anastomosis, a rare fusion of the superior and middle trunks to a 'superomiddle' trunk, a preliminary, proximal branching of the suprascapular nerve off the C5 root. We further observed an accessory 'medial anterior division' branching off the fused upper and middle trunks merging with the anterior division of the inferior trunk forming the medial cord. The latter event potentially introduced nerve fibers from C5 to C7, which are absent in common patterns. We aim to relate these observations to previous categorizations and quantifications of brachial plexus patterns. We believe that the combination of different variations in this case resulted in a unique pattern. Since this observation was made in the dissection class, we further aim to raise awareness among medical students and anatomical instructors for the likelihood of variations to textbook patterns. This will hopefully foster an appreciation of uniqueness and individuality in the interaction with future patients demonstrating that proper preparation prior to surgical interventions is always a necessary prerequisite.

The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina.

Gap junction coupling synchronizes activity among neurons in adult neural circuits, but its role in coordinating activity during development is less known. The developing retina exhibits retinal waves--spontaneous depolarizations that propagate among retinal interneurons and drive retinal ganglion cells (RGCs) to fire correlated bursts of action potentials. During development, two connexin isoforms, connexin 36 (Cx36) and Cx45, are expressed in bipolar cells and RGCs, and therefore provide a potential substrate for coordinating network activity. To determine whether gap junctions contribute to retinal waves, we compared spontaneous activity patterns using calcium imaging, whole-cell recording, and multielectrode array recording in control, single-knock-out (ko) mice lacking Cx45 and double-knock-out (dko) mice lacking both isoforms. Wave frequency, propagation speed, and bias in propagation direction were similar in control, Cx36ko, Cx45ko, and Cx36/45dko retinas. However, the spontaneous firing rate of individual retinal ganglion cells was elevated in Cx45ko retinas, similar to Cx36ko retinas (Hansen et al., 2005; Torborg and Feller, 2005), a phenotype that was more pronounced in Cx36/45dko retinas. As a result, spatial correlations, as assayed by nearest-neighbor correlation and functional connectivity maps, were significantly altered. In addition, Cx36/45dko mice had reduced eye-specific segregation of retinogeniculate afferents. Together, these findings suggest that although Cx36 and Cx45 do not play a role in gross spatial and temporal propagation properties of retinal waves, they strongly modulate the firing pattern of individual RGCs, ensuring strongly correlated firing between nearby RGCs and normal patterning of retinogeniculate projections.

Of Humans and Gerbils- Independent Diversification of Neuroligin-4 Into X- and Y-Specific Genes in Primates and Rodents.

The neural cell adhesion protein neuroligin-4 has puzzled neuroscientists and geneticist alike for almost two decades. Its clinical association with autism spectrum disorders (ASD) is well established, however, its diversification into sex chromosome-specific copies, NLGN4X and NLGN4Y, remains uncharted territory. Just recently, the presence of substantial neuroligin-4 sequence differences between humans and laboratory mice, in which Nlgn4 is a pseudoautosomal gene, could be explained as a consequence of dramatic changes affecting the pseudoautosomal region on both sex chromosomes in a subset of rodents, the clade eumuroida. In this study, we describe the presence of sex chromosome-specific copies of neuroligin-4 genes in the Mongolian gerbil (Meriones unguiculatus) marking the first encounter of its kind in rodents. Gerbils are members of the family Muridae and are closely related to mice and rats. Our results have been incorporated into an extended evolutionary analysis covering primates, rodents, lagomorphs, treeshrews and culogos comprising together the mammalian superorder euarchontoglires. We gathered evidence that substantial changes in neuroligin-4 genes have also occurred outside eumuroida in other rodent species as well as in lagomorphs. These changes feature, e.g., a general reduction of its gene size, an increase in its average GC-content as well as in the third position (GC3) of synonymous codons, and the accumulation of repetitive sequences in line with previous observations. We further show conclusively that the diversification of neuroligin-4 in sex chromosome-specific copies has happened multiple times independently during mammal evolution proving that Y-chromosomal NLGN4Y genes do not originate from a single common NLGN4Y ancestor.

RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons.

Synaptic ribbons are presynaptic specializations that define eponymous ribbon synapses. Synaptic ribbons are largely composed of RIBEYE, a protein containing an N-terminal A-domain and a carboxyterminal B-domain that is identical with CtBP2, a NAD(H)-binding transcriptional co-repressor. Previously we showed that synaptic ribbons are completely absent in RIBEYE knockout mice in which the RIBEYE A-domain-encoding exon had been deleted, but CtBP2 is still made, demonstrating that the A-domain is required for synaptic ribbon assembly. In the present study, we asked whether the RIBEYE B-domain also has an essential role in the assembly of synaptic ribbons. For this purpose, we made use of RIBEYE knockin mice in which the RIBEYE B-domain was replaced by a fluorescent protein domain, whereas the RIBEYE A-domain was retained unchanged. We found that replacing the RIBEYE B-domain with a fluorescent protein module destabilizes the resulting hybrid protein and causes a complete loss of synaptic ribbons. Our results thus demonstrate an essential role of the RIBEYE B-domain in enabling RIBEYE assembly into synaptic ribbons, reinforcing the notion that RIBEYE is the central organizer of synaptic ribbons.

Taste Receptor Activation in Tracheal Brush Cells by Denatonium Modulates ENaC Channels via Ca2+, cAMP and ACh.

Mucociliary clearance is a primary defence mechanism of the airways consisting of two components, ciliary beating and transepithelial ion transport (ISC). Specialised chemosensory cholinergic epithelial cells, named brush cells (BC), are involved in regulating various physiological and immunological processes. However, it remains unclear if BC influence ISC. In murine tracheae, denatonium, a taste receptor agonist, reduced basal ISC in a concentration-dependent manner (EC50 397 µM). The inhibition of bitter taste signalling components with gallein (Gßγ subunits), U73122 (phospholipase C), 2-APB (IP3-receptors) or with TPPO (Trpm5, transient receptor potential-melastatin 5 channel) reduced the denatonium effect. Supportively, the ISC was also diminished in Trpm5-/- mice. Mecamylamine (nicotinic acetylcholine receptor, nAChR, inhibitor) and amiloride (epithelial sodium channel, ENaC, antagonist) decreased the denatonium effect. Additionally, the inhibition of Gα subunits (pertussis toxin) reduced the denatonium effect, while an inhibition of phosphodiesterase (IBMX) increased and of adenylate cyclase (forskolin) reversed the denatonium effect. The cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh172 and the KCNQ1 potassium channel antagonist chromanol 293B both reduced the denatonium effect. Thus, denatonium reduces ISC via the canonical bitter taste signalling cascade leading to the Trpm5-dependent nAChR-mediated inhibition of ENaC as well as Gα signalling leading to a reduction in cAMP-dependent ISC. Therefore, BC activation contributes to the regulation of fluid homeostasis.

Unusually rapid evolution of Neuroligin-4 in mice.

Neuroligins (NLs) are postsynaptic cell-adhesion molecules that are implicated in humans in autism spectrum disorders because the genes encoding NL3 and NL4 are mutated in rare cases of familial autism. NLs are highly conserved evolutionarily, except that no NL4 was detected in the currently available mouse genome sequence assemblies. We now demonstrate that mice express a distant NL4 variant that rapidly evolved from other mammalian NL4 genes and that exhibits sequence variations even between different mouse strains. Despite its divergence, mouse NL4 binds neurexins and is transported into dendritic spines, suggesting that the core properties of NLs are retained in this divergent NL isoform. The selectively rapid evolution of NL4 in mice suggests that its function in the brain is under less stringent control than that of other NLs, shedding light on why its mutation in autism spectrum disorder patients is not lethal, but instead leads to a discrete developmental brain disorder.

Of mice and men - and guinea pigs?

This year marks the twentieth anniversary of the publication of the first draft of the human genome and its broad availability to the scientific community. In parallel, the annotation of the mouse genome led to the identification and analysis of countless genes by means of genetic manipulation. Today, when comparing both genomes, it might surprise that some genes are still seeking their respective homologs in either species. In this review, we aim at raising awareness for the remarkable differences between the researcher's favorite rodents, i.e., mice and rats, when it comes to the generation of rodent research models regarding genes with a particular delicate localization, namely the pseudoautosomal region on both sex chromosomes. Many of these genes are of utmost clinical relevance in humans and still miss a rodent disease model giving their absence in mice and rats or low sequence similarity compared to humans. The abundance of rodents within mammals prompted us to investigate different branches of rodents leading us to the re-discovery of the guinea pig as a mammalian research model for a distinct group of genes.