Altered salt taste response and increased tongue epithelium Scnna1 expression in adult Engrailed-2 null mice.
ABSTRACT: Sensory impairments are critical for diagnosing and characterizing neurodevelopmental disorders. Taste is a sensory modality often not well characterized. Engrailed-2 (En2) is a transcription factor critical for neural development, and mice lacking En2 (En2-/-) display signs of impaired social interaction, cognitive processes (e.g., learning and memory, conditioned fear), and neurodevelopmental alterations. As such, En2-/- mice display the behavioral deficits and neural impairments characteristic of the core symptoms associated with autism spectrum disorder (ASD). The objective of this study was to characterize the taste function in En2-/- compared with En2+/+ in adult male mice. Measuring taste responsiveness by an automated gustometer, En2 null mice had decreased lick responses for 1.6?M fructose, whereas they demonstrated an increased taste responsivity (i.e., relative to water) at 0.3?M sodium chloride and 1?M monosodium glutamate. In a separate cohort of mice, En2-/- mice had an increased preference for sodium chloride over a range of concentrations (0.032-0.3?M) compared with En2+/+ mice. Regional gene expression of the tongue epithelium demonstrated an increase in Scnn1a, T2R140, T1R3, and Trpm5 and a decrease in Pkd1l3 in En2 null mice. Taken together, such data indicate that deficits in En2 can produce sensory impairments that can have a measurable impact on taste, particularly salt taste.
Project description:Recent studies, both in vitro and in vivo, have suggested the involvement of the polycystic kidney disease-1 and -2 like genes, Pkd1l3 and Pkd2l1, in acid taste transduction. In mice, disruption of taste cells expressing PKD2L1 eliminates gustatory neural responses to acids. However, no previous data exist on taste responses in the absence of PKD1L3 or on behavioral responses in mice lacking either of these proteins. In order to assess the function of PKD1L3, we genetically engineered mice with a targeted mutation of the Pkd1l3 gene. We then examined taste responsiveness of mutant and wild-type mice using several different approaches. In separate groups of mice, we measured preference scores in 48-h 2-bottle tests, determined NaCl or citric acid taste thresholds using a conditioned taste aversion technique, and conducted electrophysiological recordings of activity in the chorda tympani and glossopharyngeal nerves. Multiple taste compounds representing all major taste qualities were used in the preference tests and nerve-recording experiments. We found no significant reduction in taste responsiveness in Pkd1l3 mutant mice in behavioral or electrophysiological tests when compared with wild-type controls. Therefore, further studies are needed to elucidate the function of PKD1L3 in taste bud cells.
Project description:Ligand-gated ion channels are important in sensory and synaptic transduction. The PKD1L3-PKD2L1 channel complex is a sour taste receptor candidate that is activated by acids. Here, we report that the proton-activated PKD1L3-PKD2L1 ion channels have the unique ability to be activated after the removal of an acid stimulus. We refer to this property as the off-response (previously described as a delayed response). Electrophysiological analyses show that acid-induced responses are observed only after the removal of an acid solution at less than pH 3.0. A small increase in pH is sufficient for PKD1L3-PKD2L1 channel activation, after exposure to an acid at pH 2.5. These results indicate that this channel is a new type of ion channel-designated as an 'off-channel'-which is activated during stimulus application but not gated open until the removal of the stimulus. The off-response property of PKD1L3-PKD2L1 channels might explain the physiological phenomena occurring during sour taste sensation.
Project description:Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.
Project description:ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.
Project description:Polycystic kidney disease 1-like 3 (Pkd1l3) is expressed specifically in sour-sensing type III taste cells that have synaptic contacts with afferent nerve fibers in circumvallate (CvP) and foliate papillae (FoP) located in the posterior region of the tongue, although not in fungiform papillae (FuP) or the palate. To visualize the gustatory neural pathways that originate from type III taste cells in CvP and FoP, we established transgenic mouse lines that express the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse Pkd1l3 gene promoter/enhancer. The WGA transgene was accurately expressed in Pkd1l3-expressing type III taste cells in CvP and FoP. Punctate WGA protein signals appeared to be detected specifically in type III taste cells but not in other types of taste cells. WGA protein was transferred primarily to a subset of neurons located in close proximity to the glossopharyngeal (GL) nerve bundles in the nodose/petrosal ganglion (NPG). WGA signals were also observed in a small population of neurons in the geniculate ganglion (GG). This result demonstrates the anatomical connection between taste receptor cells (TRCs) in the FoP and the chorda tympani (CT) nerves. WGA protein was further conveyed to neurons in a rostro-central subdivision of the nucleus of the solitary tract (NST). These findings demonstrate that the approximately 10?kb 5'-flanking region of the mouse Pkd1l3 gene functions as a type III taste cell-specific promoter/enhancer. In addition, experiments using the pkd1l3-WGA transgenic mice reveal a sour gustatory pathway that originates from TRCs in the posterior region of the tongue.
Project description:Animals use their gustatory systems to evaluate the nutritious value, toxicity, sodium content, and acidity of food. Although characterization of molecular identities that receive taste chemicals is essential, molecular receptors underlying sour taste sensation remain unclear. Here, we show that two transient receptor potential (TRP) channel members, PKD1L3 and PKD2L1, are coexpressed in a subset of taste receptor cells in specific taste areas. Cells expressing these molecules are distinct from taste cells having receptors for bitter, sweet, or umami tastants. The PKD2L1 proteins are accumulated at the taste pore region, where taste chemicals are detected. PKD1L3 and PKD2L1 proteins can interact with each other, and coexpression of the PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Finally, PKD1L3 and PKD2L1 are activated by various acids when coexpressed in heterologous cells but not by other classes of tastants. These results suggest that PKD1L3 and PKD2L1 heteromers may function as sour taste receptors.
Project description:Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.
Project description:Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca(2+) imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca(2+) increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.
Project description:Intrauterine ischemia-hypoxia is detrimental to the developing brain and leads to white matter injury (WMI), encephalopathy of prematurity (EP), and often to cerebral palsy (CP), but the related pathophysiological mechanisms remain unclear. In prior studies, we used mild intrauterine hypoperfusion (MIUH) in rats to successfully reproduce the diversity of clinical signs of EP, and some CP symptoms. Briefly, MIUH led to inflammatory processes, diffuse gray and WMI, minor locomotor deficits, musculoskeletal pathologies, neuroanatomical and functional disorganization of the primary somatosensory and motor cortices, delayed sensorimotor reflexes, spontaneous hyperactivity, deficits in sensory information processing, memory and learning impairments. In the present study, we investigated the early and long-lasting mechanisms of pathophysiology that may be responsible for the various symptoms induced by MIUH. We found early hyperreflexia, spasticity and reduced expression of KCC2 (a chloride cotransporter that regulates chloride homeostasis and cell excitability). Adult MIUH rats exhibited changes in muscle contractile properties and phenotype, enduring hyperreflexia and spasticity, as well as hyperexcitability in the sensorimotor cortex. Taken together, these results show that reduced expression of KCC2, lumbar hyperreflexia, spasticity, altered properties of the soleus muscle, as well as cortical hyperexcitability may likely interplay into a self-perpetuating cycle, leading to the emergence, and persistence of neurodevelopmental disorders (NDD) in EP and CP, such as sensorimotor impairments, and probably hyperactivity, attention, and learning disorders.
Project description:Mutations in polycystin proteins PKD1 and TRPP2 lead to autosomal dominant polycystic kidney disease. These two proteins form a receptor-ion channel complex on primary cilia. PKD1 undergoes an autoproteolysis at the N terminal G-protein-coupled receptor proteolytic site (GPS), which is essential for the function of PKD1. Whether GPS cleavage happens in other PKD proteins and its functional consequence has remained elusive. Here we studied the GPS cleavage of PKD1L3, a protein that associates with TRPP3 in taste cells and may play a role in sour taste. Our results show that PKD1L3 also undergoes GPS cleavage. Mutation at the GPS abolishes the cleavage, and the non-cleavable mutant does not traffic to the plasma membrane when associated with TRPP3. We also found that a splice variant of PKD1L3, which was originally identified in taste buds, is not cleaved. Amino acids L708 and S709, which are missing in this splice variant, are crucial for the GPS cleavage of PKD1L3 and the trafficking of the PKD1L3/TRPP3 complex. Our results gain insight into the molecular mechanism of the GPS cleavage of PKD1L3. The presence of the non-cleavable variant suggests the potential in vivo function of uncleaved PKD proteins.