A large increase of sour taste receptor cells in Skn-1-deficient mice does not alter the number of their sour taste signal-transmitting gustatory neurons.

The connections between taste receptor cells (TRCs) and innervating gustatory neurons are formed in a mutually dependent manner during development. To investigate whether a change in the ratio of cell types that compose taste buds influences the number of innervating gustatory neurons, we analyzed the proportion of gustatory neurons that transmit sour taste signals in adult Skn-1a-/- mice in which the number of sour TRCs is greatly increased. We generated polycystic kidney disease 1 like 3-wheat germ agglutinin (pkd1l3-WGA)/Skn-1a+/+ and pkd1l3-WGA/Skn-1a-/- mice by crossing Skn-1a-/- mice and pkd1l3-WGA transgenic mice, in which neural pathways of sour taste signals can be visualized. The number of WGA-positive cells in the circumvallate papillae is 3-fold higher in taste buds of pkd1l3-WGA/Skn-1a-/- mice relative to pkd1l3-WGA/Skn-1a+/+ mice. Intriguingly, the ratio of WGA-positive neurons to P2X2-expressing gustatory neurons in nodose/petrosal ganglia was similar between pkd1l3-WGA/Skn-1a+/+ and pkd1l3-WGA/Skn-1a-/- mice. In conclusion, an alteration in the ratio of cell types that compose taste buds does not influence the number of gustatory neurons that transmit sour taste signals.

Expression of serotonin receptor genes in cranial ganglia.

Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells.

Amino acid substitutions in PB1 of avian influenza viruses influence pathogenicity and transmissibility in chickens.

UNLABELLED: Amino acid substitutions were introduced into avian influenza virus PB1 in order to characterize the interaction between polymerase activity and pathogenicity. Previously, we used recombinant viruses containing the hemagglutinin (HA) and neuraminidase (NA) genes from the highly pathogenic avian influenza virus (HPAIV) H5N1 strain and other internal genes from two low-pathogenicity avian influenza viruses isolated from chicken and wild-bird hosts (LP and WB, respectively) to demonstrate that the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in chickens is regulated by the PB1 gene (Y. Uchida et al., J. Virol. 86:2686-2695, 2012, doi:http://dx.doi.org/10.1128/JVI.06374-11). In the present study, we introduced a C38Y substitution into WB PB1 and demonstrated that this substitution increased both polymerase activity in DF-1 cells in vitro and the pathogenicity of the recombinant viruses in chickens. The V14A substitution in LP PB1 reduced polymerase activity but did not affect pathogenicity in chickens. Interestingly, the V14A substitution reduced viral shedding and transmissibility. These studies demonstrate that increased polymerase activity correlates directly with enhanced pathogenicity, while decreased polymerase activity does not always correlate with pathogenicity and requires further analysis. IMPORTANCE: We identified 2 novel amino acid substitutions in the avian influenza virus PB1 gene that affect the characteristics of highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype, such as viral replication and polymerase activity in vitro and pathogenicity and transmissibly in chickens. An amino acid substitution at residue 38 in PB1 directly affected pathogenicity in chickens and was associated with changes in polymerase activity in vitro. A substitution at residue 14 reduced polymerase activity in vitro, while its effects on pathogenicity and transmissibility depended on the constellation of internal genes.

Differential expression analysis throughout the weaning period in the mouse cerebral cortex.

At weaning, mammals switch from drinking mother's milk to eating foods of environmental origin. These foods contain natural compounds with novel tastes and textures, which are provided to the young for the first time following the termination of breastfeeding. This novel eating experience may alter the cognitive brain function of mammalian babies, increasing their reactions to their food environments. Because the cerebral cortex is a central organ for cognition and learning, we investigated differences in whole-gene expression profiles in the mouse cerebral cortex using microarray analysis before and after weaning. Of 45,037 murine genes, 35 genes were upregulated and 31 genes were downregulated, in response to weaning. In particular, immediate early genes, molecular chaperones, and myelin-related genes were upregulated. In situ hybridization analysis revealed that the mRNA for an immediate early gene, Egr-2/KROX-20, was transported from the nucleus to the cell body at layer 5/6 of the somatosensory cortex during weaning. In contrast, in animals without any food supply other than mother's milk, Egr-2/KROX-20 mRNA was retained within the nucleus at the somatosensory cortex. These data suggest that the novel experience of food intake modulates gene expression profiles in the murine cerebral cortex at the weaning stage.

Genetic tracing of the neural pathway for bitter taste in t2r5-WGA transgenic mice.

To visualize the neural pathways originating from bitter taste receptor cells (TRCs), we generated transgenic mice expressing the transneuronal tracer wheat germ agglutinin (WGA) under the control of the mouse T2R5 gene promoter/enhancer (t2r5-WGA mice). WGA mRNA was specifically expressed in bitter TRCs. The WGA protein was detected in bitter TRCs and nerve processes in taste buds, but not in sweet, umami, or sour TRCs. The WGA protein was transferred to a subset of sensory neurons in the geniculate and nodose/petrosal ganglia. These results suggest that bitter TRCs, which are devoid of synaptic structures, are innervated by gustatory neurons and that bitter sensory information is directly transmitted to specific gustatory neurons. The t2r5-WGA mice provide a useful tool for identifying gustatory relay neurons in the peripheral sensory ganglia responsible for aversive sensations.

Spatial differences in molecular characteristics of the pontine parabrachial nucleus.

Neurons in the pontine parabrachial nucleus (PBN) transduce signals for the general visceral sensory, somatic sensory, gustatory, and autonomic nervous systems, and the various PBN neurons that perform these functions are intermingled. In this study, we analyzed PBN gene expression profiles in male Wistar rats and obtained data on gene expression in the PBN and the principal sensory nucleus of the trigeminal nerve (Pr5). Using these data in combination with in situ hybridization analyses, we identified genes that showed higher expression in the PBN than in Pr5. Our findings indicate that expression patterns in the PBN were different for different genes: Fxyd6, syt5, and plxnc1 were expressed in many neuron populations in the PBN, while the expression patterns of calcr and asb4 were restricted to the central lateral subnucleus and waist area. Furthermore, calcr and asb4 expression patterns were distinct from those of neurotransmitters/neuropeptides such as neurotensin and calcitonin gene-related peptides. Satb2 was specifically expressed in the waist area, which is essential for gustation. In-depth analysis of spatial distribution in the PBN enabled classification of the genes into seven characteristic spatial expression patterns. Expression signatures differed significantly in the subnuclei of the rostral half, mediodorsal half, and ventrolateral third of the PBN, indicating a correlation between the spatial arrangement of the subnuclei and the molecular characteristics of the corresponding neurons. Thus, our results provide valuable information regarding the molecular features and neurotransmission mechanisms of PBN neurons that transmit specific types of information.

Functional organization of the rodent parabrachial nucleus.

The rodent parabrachial nucleus (PBN) is not merely a sensory relay station but also plays an important role in integrating various ascending and descending inputs together with plastic changes of neuronal responses after learning and experience. The limbic and reward systems receive ingestion-related information via the cortical areas in primates, whereas in rodents the information is sent to these systems mostly via the PBN. To explore how the rat PBN is functionally organized, we detected activation patterns of neurons mainly by means of c-fos immunohistochemistry to show neuronal activation in different situations of ingestive behavior. The expression pattern was different under nutritionally replete and deficient conditions, perceptually new and familiar conditions, and learned and unlearned conditions. As for the possible functions, the rostral part of the external lateral subnucleus is related to general visceral inputs; the caudal part of the external lateral subnucleus, aversive behavior; the dorsal lateral subnucleus, ingestive behavior; and the central medial subnucleus, taste of NaCl. Because several genes were localized in specific subnuclei, we are trying to correlate the gene expressions with possible functional significance.