Takatoshi Nagai

Differential blocking effects of a spider toxin on synaptic and glutamate responses in the afferent synapse of the acoustico-lateralis receptors of Plotosus.

The hypothesis that glutamate is the afferent transmitter in the acoustico-lateralis receptors was examined in Plotosus electroreceptors. JSTX , a spider toxin known to specifically block glutamate receptors, irreversibly abolished afferent impulse discharges induced by iontophoretically applied glutamate, whereas those induced synaptically by focal stimulation of receptor cells were little affected. Such differential blocking effects by JSTX , complementary to other biochemical data, further provide pharmacological evidence against the glutamate hypothesis.

Desert toads discriminate salt taste with chemosensory function of the ventral skin.

Toads obtain water by absorption across their skin. When dehydrated, desert toads exhibit stereotyped hydration behavior in which they press their ventral skin onto a moist surface. However, dehydrated toads avoid surfaces moistened with hyperosmotic NaCl and KCl solutions (Hoff KvS, Hillyard SD. 1993. J. Exp. Biol. 183:347–351). We have studied neural mechanisms for this avoidance with physiologic, behavioral, and morphologic approaches. Spinal nerves innervating the ventral skin could be stimulated by exposure to a hyperosmotic NaCl solution applied to the outer surface of the skin. This neural response occurred with much longer latency than to mechanical stimulation and could be reduced by amiloride, a blocker for Na+ channels known to be responsible for epithelial ion transport and salt taste transduction. In behavioral experiments, avoidance of a NaCl solution was also reduced by adding amiloride to the solution, suggesting involvement of amiloride‐sensitive Na+ channels for detecting the hyperosmotic salt solution. Neural tracing with fluorescent dye revealed spinal nerve endings and connections to putative receptor cells, both located in the deeper layer of the epidermis. Either of these or both may be associated with the transduction of Na+ flowing into the skin. The ability of toads to detect hyperosmotic salt solutions in their environment reveals a previously unknown chemosensory function for spinal nerves in anuran amphibians. J. Comp. Neurol. 408:125–136, 1999.

Electrophysiological and behavioral studies of taste discrimination in the axolotl (ambystoma mexicanum)

Electrophysiological and behavioral experiments were performed to determine whether the taste system of the aquatic salamander, axolotl, discriminates taste stimuli. Taste responses were recorded extracellularly from the glossopharyngeal nerve bundle. The behavioral responses of axolotls towards various concentrations of NaCl, KCl, citric acid, quinine-hydrochloride, and sucrose were quantified by measuring the ratio of rejection towards gel pellets, each containing either unitary stimuli or binary mixtures of these chemicals. Rejection ratios [rejection/(rejection+swallowing)] towards the unitary stimuli except sucrose increased with concentration, but were not a single function of the magnitude of neural response induced by the stimuli. Degree of rejection was different depending on the quality of taste stimuli, suggesting that information processing of taste quality occurs in axolotls. The potential of NaCl to induce positive feeding behavior (swallowing) was suggested by a reduction in the rejection ratio of quinine-tainted pellets when they were mixed with 100 mM NaCl. Differential behavioral responses to quinine and NaCl show that axolotls have the ability to discriminate the taste quality of these stimuli.

Ultrastructure of Merkel-like cells labeled with carbocyanine dye in the non-taste lingual epithelium of the axolotl

Fluorescent carbocyanine dye (diI), applied to the glossopharyngeal (IX) nerve of the axolotl, transneuronally labeled solitary cells in the non-taste lingual epithelium. With diaminobenzidine (DAB), the diI was photoconverted to a dark, electron-dense product. The labeled cell had a large nucleus with invaginations, dense-cored vesicles in the cytoplasm, and finger-like processes. These are reminiscent of morphological features of cutaneous Merkel cells, suggesting that solitary cells innervated by the IX nerve are associated with mechanosensory function of the IX nerve system.

Localization of Water Channels in the Skin of Two Species of Desert Toads, Anaxyrus (Bufo) punctatus and Incilius (Bufo) alvarius

Anuran amphibians obtain water by osmosis across their ventral skin. A specialized region in the pelvic skin of semiterrestrial species, termed the seat patch, contains aquaporins (AQPs) that become inserted into the apical plasma membrane of the epidermis following stimulation by arginine vasotocin (AVT) to facilitate rehydration. Two AVT-stimulated AQPs, AQP-h2 and AQP-h3, have been identified in the epidermis of seat patch skin of the Japanese tree frog, Hyla japonica, and show a high degree of homology with those of bufonid species. We used antibodies raised against AQP-h2 and AQP-h3 to characterize the expression of homologous AQPs in the skin of two species of toads that inhabit arid desert regions of southwestern North America. Western blot analysis of proteins gave positive results for AQP-h2-like proteins in the pelvic skin and also the urinary bladder of Anaxyrus (Bufo) punctatus while AQP-h3-like proteins were found in extracts from the pelvic skin and the more anterior ventral skin, but not the urinary bladder. Immunohistochemical observations showed both AQP-h2- and AQP-h3-like proteins were present in the apical membrane of skin from the pelvic skin of hydrated and dehydrated A. punctatus. Further stimulation by AVT or isoproterenol treatment of living toads was not evident. In contrast, skin from hydrated Incilius (Bufo) alvarius showed very weak labeling of AQP-h2- and AQP-h3-like proteins and labeling turned intense following stimulation by AVT. These results are similar to those of tree frogs and toads that occupy mesic habitats and suggest this pattern of AQP expression is the result of phylogenetic factors shared by hylid and bufonid anurans.

The glossopharyngeal nerve of the axolotl labeled with carbocyanine dye (diI)

Fluorescent carbocyanine dye (diI) was used to label the glossopharyngeal (IX) nerve in the fixed preparation of the Mexican salamander, axolotl. When the cell bodies were viewed with a confocal laser scanning microscope and Nomarski optics, the cytoplasm was brightly fluorescent, but not the cell nucleus. The cell bodies which send peripheral axons in the two branches of the IX nerve were mainly distributed in the rostral part of the combined glossopharyngeal-vagus ganglion, but a few cells were also distributed in the middle and caudal parts. This may indicate a relatively undifferentiated organization of the IX nerve in the ganglion.

Taste Discrimination in Salamanders

Salamanders are excellent experimental models for studying cellular mechanisms of taste reception because their taste receptor cells are large and form large taste buds of simple structure [1–3]. Intracellular recordings were made from taste receptor cells of the mudpuppy Necturus maculosus [4–7] and the tiger salamander Ambystoma tigrinum [8,9]. The chemosensitivity of the glossopharyngeal nerve (nIX) innervating a large area of the salamander lingual epithelium was studied in the mudpuppy [10,11] and the Mexican salamander Ambystoma mexicanum [12]. The ultrastructure of taste cells and associated synapses was studied in the mudpuppy [2,13,14] and the Mexican salamander [1,3]. Although there has been a wealth of physiological and anatomical studies, behavioral studies of the salamander’s ability to discriminate taste are few [15,16]. Therefore we designed behavioral experiments to examine if salamanders can discriminate the chemicals that are commonly used in physiological experiments as taste stimuli.

Artificial Neural Networks Analyze Response Patterns of Cortical Taste Neurons in the Rat

For many years the similarity of taste stimuli has been measured mainly by the determination of a correlation coefficient across the neurons activated by the stimuli. In a previous study, we developed a new method for analyzing gustatory neural activities, using artificial neural networks [1]. In the present study, we examined further the operation of these networks in regard to neural responses induced by a variety of taste stimuli, and the effect of “pruning” on the network output. Three-layer neural networks were trained by the back-propagation learning algorithm to classify the neural response patterns to the four basic taste qualities (1.0 M sucrose, 0.03 M HCl, 0.01 M quinine HCl, and 0.1 M NaCl). The networks had four output units representing the four basic taste qualities: sweet (S), sour (H), bitter (Q), and salt (N). The input units represented rat cortical neurons from which the response patterns (impulses/3 s) were recorded in a separate physiological experiment [2]. After training, the response patterns to test stimuli (0.02 M sodium saccharin, 0.3 M KCl, 0.3 M MgCl2, 0.1 M NaNO3, 0.01 M tartaric acid, 0.3 M CaCl2, 0.1 M monosodium L-glutamate [MSG] and 0.1 M inosine 5′-monophosphate [IMP]) were presented to the input units. For NaNO3, the networks produced large outputs (around 0.9) almost exclusively in the N unit, showing pure salt taste in the stimulus. Large and exclusive outputs were also produced in the H unit for KCl (around 0.7) and in the Q unit for tartaric acid (around 0.5). On the other hand, outputs suggesting mixed bitter and salt tastes were produced for CaCl2 and MgCl2. As to the similarity of the test stimuli to the four basic taste qualities, the neural networks presented a clearer and more definite result than the conventional correlation analysis. For MSG and IMP, the networks produced exclusive but small outputs (around 0.2) in the N unit, although some investigators classify these stimuli as an umami taste that is independent of the four basic taste qualities.