5-HT3A -driven green fluorescent protein delineates gustatory fibers innervating sour-responsive taste cells: A labeled line for sour taste?

Taste buds contain multiple cell types with each type expressing receptors and transduction components for a subset of taste qualities. The sour sensing cells, Type III cells, release serotonin (5-HT) in response to the presence of sour (acidic) tastants and this released 5-HT activates 5-HT3 receptors on the gustatory nerves. We show here, using 5-HT3A GFP mice, that 5-HT3 -expressing nerve fibers preferentially contact and receive synaptic contact from Type III taste cells. Further, these 5-HT3 -expressing nerve fibers terminate in a restricted central-lateral portion of the nucleus of the solitary tract (nTS)-the same area that shows increased c-Fos expression upon presentation of a sour tastant (30 mM citric acid). This acid stimulation also evokes c-Fos in the laterally adjacent mediodorsal spinal trigeminal nucleus (DMSp5), but this trigeminal activation is not associated with the presence of 5-HT3 -expressing nerve fibers as it is in the nTS. Rather, the neuronal activation in the trigeminal complex likely is attributable to direct depolarization of acid-sensitive trigeminal nerve fibers, for example, polymodal nociceptors, rather than through taste buds. Taken together, these findings suggest that transmission of sour taste information involves communication between Type III taste cells and 5-HT3 -expressing afferent nerve fibers that project to a restricted portion of the nTS consistent with a crude mapping of taste quality information in the primary gustatory nucleus.

Differential localization of NT-3 and TrpM5 in glomeruli of the olfactory bulb of mice.

Olfactory sensory neurons that express transient receptor potential channel M5 (TrpM5) or neurotrophin-3 (NT-3) project to defined clusters of glomeruli situated ventrally in the main olfactory bulb. Using genetically labeled mice, we investigated whether expression of NT-3-driven ßgal and TrpM5-driven GFP marked overlapping sets of glomeruli and whether expression of these markers was coordinated. Our results indicate that these markers largely characterize independent sets of olfactory sensory neuron axons and glomeruli. Further, in glomeruli in which both TrpM5-GFP and NT-3-ßgal labeled axons occur, they are expressed independently. The nature of staining for these two markers also differs within glomeruli. Within each labeled TrpM5-positive glomerulus, the level of TrpM5-GFP expression was similar throughout the glomerular neuropil. In contrast, NT-3-driven ßgal expression levels are heterogeneous even within heavily labeled glomeruli. In addition, a population of very small TrpM5-GFP positive glomeruli is apparent while no similar populations of NT-3-ßgal glomeruli are evident. Taken together, these data suggest that TrpM5 and NT-3 characterize two largely independent receptor populations both conveying odorant information to the ventral olfactory bulb.

"Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.

Taste buds contain a variety of morphological and histochemical types of elongate cells. Serotonin, neuron-specific enolase (NSE), ubiquitin carboxyl terminal hydrolase (PGP 9.5), and neural cell adhesion molecule (N-CAM) all have been described as being present in the morphologically defined Type III taste cells in rats. In order to determine whether these substances coexist in a single cell, we undertook immunohistochemical and ultrastructural analysis of taste buds in rats. Double-label studies show that PGP 9.5 and NSE always colocalize. In contrast, PGP 9.5 and serotonin seldom colocalize. Further, whereas the serotonin-immunoreactive cells are always slender and elongate, the PGP 9.5/NSE population comprise two morphological types--one slender, the other broader and pyriform. Although gustducin-immunoreactive taste cells appear similar in overall shape to the pyriform PGP 9.5/NSE population, gustducin never colocalizes with PGP 9.5 or NSE. The serotonin-immunoreactive taste cells have an invaginated nucleus, synaptic contacts with nerve fibers, and taper apically to a single, large microvillus. These are all characteristics of Type III taste cells described previously in rabbits (Murray [1973] Ultrastructure of Sensory Organs I. Amsterdam: North Holland. p 1-81). PGP 9.5-immunoreactive taste cells exhibit two morphological varieties. One type is similar to the serotonin-immunoreactive population, containing an invaginated nucleus, synapses with nerve fibers, and a single large microvillus. The other type of PGP 9.5-immunoreactive taste cell has a large round nucleus and the apical end of the cell tapers to a tuft of short microvilli, which are characteristics of Type II taste cells. Thus, in rats, some Type III cells accumulate serotonin but do not express PGP 9.5, whereas others express PGP 9.5 but do not accumulate amines. Similarly, Type II taste cells come in at least two varieties: those immunoreactive for gustducin and those immunoreactive for PGP 9.5.

Maintenance of rat taste buds in primary culture.

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.

Variability of position of the P2 glomerulus within a map of the mouse olfactory bulb.

Olfactory receptor neurons (ORNs) that express a common odorant receptor molecule target specific glomeruli in the olfactory bulb. We systematically assessed the location of the olfactory glomeruli that receive input from ORNs expressing P2 receptors in the P2-internal ribosome entry site-tau-lacZ mouse. We present a new mapping method that includes an Internet-accessible computer program for generating two- and three-dimensional maps of the glomerular sheet in the olfactory bulbs of mice. Cylindrical coordinates were used to define glomerular location: The coordinates were given as the anteroposterior (AP) distance parallel to the long axis of the bulb (rostrocaudal; RC) and angular measurements with origin defined by the remnant ependymal layer in the center of the granule cell layer in the bulb. Using this method, we can apply rigorous statistical methods to give objective estimates of position and variability. At the 95% confidence interval, the lateral P2 glomerulus lies at coordinates 1,008 microm +/- 306 microm AP x 146 degrees +/- 12 degrees, and the medial P2 glomerulus lies at 1,828 microm +/- 196 microm AP x 204 degrees +/- 8 degrees. We estimate that these coordinates encompass a domain containing 29 and 37 of the 1,800 glomeruli ( approximately 2%) for the lateral and medial glomeruli, respectively. Furthermore, the data reported here demonstrate that the rostrocaudal position of small P2 glomeruli is three times more variable than that of large glomeruli.

Kainate-activated cobalt uptake in the primary gustatory nucleus in goldfish: visualization of the morphology and distribution of cells expressing AMPA/kainate receptors in the vagal lobe.

Gustatory afferent fibers of the vagus nerve that innervate taste buds of the oropharynx of the goldfish, Carassius auratus, project to the vagal lobe, which is a laminated gustatory nucleus in the dorsal medulla. As in the mammalian gustatory system, responses by second-order cells in the goldfish medulla are mediated by N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic glutamate receptors. We utilized a cobalt uptake technique to label vagal lobe neurons that possess cobalt-permeable ionotropic glutamate receptors. Vagal lobe slices were bathed in kainate (40 microM) or glutamate (0.5 or 1 mM) in the presence of CoCl(2), which can pass into cells through the ligand-gated cation channels of non-NMDA receptors made up of certain subunit combinations. Cobalt-filled cells and dendrites were observed in slices that were activated by kainate or glutamate, but not in control slices that were bathed in CoCl(2) alone, nor in slices that were bathed with the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (10 microM) in addition to an agonist. Likewise, simple depolarization of the cells with KCl failed to induce cobalt loading. Cobalt-filled round unipolar cells, elongate or globular bipolar cells, and multipolar cells with elongate or polygonal perikarya were distributed throughout the cell layers in the sensory zone of the vagal lobe. Numerous labeled neurons had dendrites spanning layers IV and VI, the two principal layers of primary afferent input. Apical and basal dendrites often extended radially through neighboring laminae, but many cells also extended dendrites tangential to the lamination of the sensory zone. In the motor layer, cell bodies and proximal dendrites of small, multipolar neurons, and large motoneurons were regularly loaded with cobalt.

Mature olfactory receptor neurons express connexin 43.

The expression of the gap junction subunit connexin 43 was studied in the olfactory epithelium of adult mice. In agreement with conclusions from previous immunohistochemical studies, we observed expression of mRNA encoding for connexin 43 in layers of the epithelium containing nuclei belonging to sustentacular cells. However, we also observed expression of connexin 43 mRNA in the layers containing nuclei belonging to mature olfactory receptor neurons (ORNs), immature ORNs, and basal cells. Connexin 43 mRNA expression was low in dorsomedial regions of the nasal cavity but higher ventrally. This differential regional distribution was consistent with expression in a transgenic mouse of a LacZ reporter gene driven by the proximal 6.5 kb of the connexin 43 promoter. LacZ was expressed in cells colabeled with antibody against olfactory marker protein (OMP), corroborating that mature ORNs express connexin 43. LacZ staining also was observed in sustentacular and basal cells and in immature ORNs. Double-label studies with antibodies against connexin 43 and OMP and expression of connexin 43 in the epithelium of bulbectomized mice were also consistent with expression of connexin 43 in mature ORNs. This is the first report of expression of a connexin subunit in mature ORNs. Our findings of connexin subunits in mature ORNs raise the novel possibility that gap junctions may play a fundamental role in information processing in the olfactory epithelium.

Phyletic distribution of crypt-type olfactory receptor neurons in fishes.

The olfactory epithelium of teleost fishes contains ciliated and microvillous olfactory receptor neurons intermingled with supporting cells. Recently the crypt cell, a third type of olfactory receptor neuron (ORN), was described for two ostariophysans. This type of ORN bears apical microvilli as well as occult cilia extending into a crypt at the apex of the cell. The present study used scanning and transmission electron-microscopic methods to examine how widespread this cell type is in other groups of fish. We investigated the olfactory epithelia of 18 species, freshwater and marine, including various actinopterygian fish as well as 2 species of lungfishes belonging to the sarcopterygians. Crypt cells were detected in 13 species of actinopterygian fish, but in none of the sarcopterygian lungfishes. Crypt cells are present in basic as well as in highly derived actinopterygians. We conclude that crypt cells are a common feature of actinopterygian fish.

Ascending spinal systems in the fish, Prionotus carolinus.

The fin rays of the pectoral fin of the sea robins (teleostei) are specialized chemosensory organs heavily invested with solitary chemoreceptor cells innervated only by spinal nerves. The rostral spinal cord of these animals is marked by accessory spinal lobes which are unique enlargements of the dorsal horn of the rostral spinal segments receiving input from the fin ray nerves. Horseradish peroxidase (HRP) and 1,1;-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (diI) were used as anterograde and retrograde tracers to examine the connectivity of these accessory lobes and the associated ascending spinal systems in the sea robin, Prionotus carolinus. The majority of dorsal root fibers terminate within the accessory lobes at or nearby their level of entrance into the spinal cord. A few dorsal root axons turn rostrally in the dorsolateral fasciculus to terminate in the lateral funicular complex situated at the spinomedullary junction. The lateral funicular complex also receives a heavy projection from the ipsilateral accessory lobes. In addition, it contains a few large neurons that project back onto the accessory lobes. Injections of either diI or HRP into the lateral funicular complex label fibers of the medial lemniscus which crosses the midline in the caudal medulla to ascend along the ventral margin of the contralateral rhombencephalon. Within the medulla, fibers leave the medial lemniscus to terminate in the inferior olive and in the ventrolateral medullary reticular formation. Upon reaching the midbrain, the medial lemniscus turns dorsally to terminate heavily in a lateral division of the torus semicircularis, in the ventral optic tectum, and in the lateral subnucleus of the nuc. preglomerulosus of the thalamus. Lesser projections also reach the posterior periventricular portion of the posterior tubercle with a few fibers terminating along the ventral, posterior margin of the ventromedial (VM) nucleus of the thalamus. The restricted projection to the ventral tectum is noteworthy in that this part of the tectum maintains the representation of the ventral visual field, that is, the area in which the fin rays lie. A prominent spinocerebellar system is also evident. Both direct and indirect spinocerebellar fibers can be followed through the dorsolateral fasciculus, with or without relay in the lateral funicular nucleus and terminating in a restricted portion of the granule cell layer of the ipsilateral corpus cerebelli. The similarities in connectivity of the spinal cord between the sea robins and other vertebrates are striking. It is especially notable because sea robins utilize the chemosensory input from the fin rays to localize food in the environment. Thus, although these fish use their spinal chemosense as other fishes use their external taste systems, the spinal chemosense apparently relies on the medial lemniscal system to guide this chemically driven feeding behavior.

Distribution of trigeminal fibers in the primary facial gustatory center of channel catfish, Ictalurus punctatus.

Previous studies in several fishes including catfish, have shown that primary trigeminal nerve (NV) axons terminate not only in the principal and spinal trigeminal nuclei, but in the facial (gustatory) lobes. The present study was undertaken to determine the extent and distribution of trigeminal terminations within the facial lobe (FL) and principal trigeminal nucleus (nVpr) in the channel catfish, Ictalurus punctatus. In order to reveal the distribution of trigeminal fibers, the carbocyanine dye, diI, was applied to the central cut stump of the trigeminal root in isolated, paraformaldehyde-fixed brains. After a diffusion period of 10-90 days, the brains were serially sectioned on a vibratome and examined with epifluorescence. The trigeminal motor nucleus (nVm) and principal sensory nucleus lie near the level of entrance of NV. The majority of primary trigeminal fibers, however, sweep caudally after entering into the brain to form the descending root. At the level of the caudal third of the FL, collaterals emitted by the descending root fibers turn medially and dorsally to terminate in the FL. The trigeminal fibers are coarser than the facial nerve (NVII) fibers which terminate within the same structure. The trigeminal fibers terminate throughout the FL except for the lateral-most lobule which contains the representation of taste buds innervated by the recurrent branch of NVII, i.e., those over the trunk and tail of the animal. These results show that in catfish, the trigeminal input to the primary gustatory complex is restricted to those portions of the nucleus receiving chemosensory inputs from the face and barbels, i.e., the trigeminally innervated sensory fields.

NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish.

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.

Expression of sonic hedgehog, patched, and Gli1 in developing taste papillae of the mouse.

Lingual taste buds form within taste papillae, which are specialized structures that develop in a characteristic spatial and temporal pattern. To investigate the signaling events responsible for patterning and morphogenesis of taste papillae, the authors examined the time course and distribution of expression of several related developmental signaling genes as well as the time course of innervation of taste papillae in mouse embryos from embryonic day 12 (E12) to E18. Lingual expression of the signaling molecule Sonic hedgehog (Shh), its receptor Patched (Ptc), and the Shh-activated transcription factor Gli1 were assayed by using in situ hybridization. Shh is expressed broadly in the lingual epithelium at E12 but becomes progressively restricted to developing circumvallate and fungiform papillary epithelia. Shh is expressed specifically within the central cells of the papillary epithelium starting at E13.5 and persisting through E18. Ptc and Gli1 expression follow a pattern similar to that of Shh. Compared with Shh, Ptc is expressed in larger regions surrounding the central papillary cells and also in the mesenchyme underlying Shh-expressing epithelium. Innervation of taste papillae was examined by using the panneuronal antibody to ubiquitin carboxyl terminal hydrolase (protein gene product 9.5). Nerves reach the basal lamina of developing taste papillae at E14 to densely innervate the papillary epithelium by E16. Thus, the pattern of Shh expression within developing taste papillae is established prior to innervation, ruling out neuronal induction of papillae. The results suggest that the Shh signaling pathway may be involved in: 1) establishing papillary boundaries in taste papilla morphogenesis, 2) papillary epithelial-mesenchymal interactions, and/or 3) specifying the location or development of taste buds within taste papillae.

Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone.

Amiloride-sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit-specific antibodies directed against alpha, beta, and gamma subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for beta and gamma subunits. Dual localization experiments showed that immunoreactivity for the taste cell-specific G protein, gustducin, occurs in a subset ofrENaC positive taste cells. Aldosterone is known to increase the amiloride sensitivity of the NaCl taste response. In our study, increases in blood aldosterone levels enhanced the intensity of apical immunoreactivity for beta and gamma rENaC in taste cells of all papillae. In addition, whole cell recordings from isolated taste cells showed that in fungiform papillae, aldosterone increased the number of amiloride-sensitive taste cells and enhanced the current amplitude. In vallate taste cells, which are normally unresponsive to amiloride, aldosterone treatment induced an amiloride sensitive current in about half of the cells. Immunoreactivity for rENaC subunits also was present in nonsensory epithelial cells, especially in the anterior portion of the tongue. In addition, immunoreactivity for all subunits, but especially beta and gamma, was associated with some nerve fibers innervating taste papillae. These extragustatory sites of rENaC expression may indicate a role for this channel in paracellular transduction of sodium ions.

Differential projections of ciliated and microvillous olfactory receptor cells in the catfish, Ictalurus punctatus.

The primary olfactory projections of channel catfish Ictalurus punctatus have been examined with postmortem tracing by using either 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate or 1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). Following DiI deposition into small areas in different parts of the posterior olfactory bulb, olfactory sensory neurons always were labeled throughout the olfactory epithelium. No obvious topographical mapping exists between the epithelium and olfactory bulb. The different dye placements, however, did result in labeling of different morphologies of receptor cells, depending on the site of injection. Retrogradely labeled neurons in the olfactory epithelium were classified into three types on the basis of their height: tall, intermediate, and short. Tall olfactory sensory neurons had perikarya at the bottom one-fourth of the epithelium, extended slender dendrites to the epithelial surface, and possessed numerous cilia on the apical dendritic tips. These tall olfactory sensory neurons were labeled predominantly following DiI applications to the ventral part of the posterior olfactory bulb. In contrast, the short olfactory sensory neurons had perikarya situated within the superficial half of the epithelium and with short apical dendrites bearing microvilli. These short olfactory sensory neurons projected predominantly to the dorsal, posterior olfactory bulb. Thus, short microvillous receptor cells and tall ciliated receptor cells connect to different parts of the olfactory bulb, although the receptor cells are intermingled within the olfactory epithelium. Because different parts of the olfactory bulb are thought to respond preferentially to different classes of odorants, these results suggest that receptor cell morphology may be related to odorant quality detection. In addition, to compare this study with previous in vivo studies, Fluoro-Gold was injected in vivo into either the olfactory bulb or intraperitoneally. These in vivo studies show that so-called "type II ciliar receptor cells" of the nonsensory epithelium are labeled nonselectively by blood-borne substances, but they are not labeled by postmortem injections of DiI anywhere in the olfactory bulb.

The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.

Excitatory amino acid neurotransmission in the primary gustatory nucleus of the goldfish Carassius auratus.

The vagal lobe in goldfish is a laminated structure in the midmedulla responsible for processing vagal gustatory input from the oropharynx. The anatomical arrangement of the vagal lobe is conducive to an in vitro slice preparation for investigating the physiology and pharmacology of primary gustatory fibers. Postsynaptic population responses (N2 and N3) were evoked from sensory layers of the vagal lobe following stimulation of the incoming vagal fibers. Application of 100 microM kynurenic acid, a broad spectrum glutamate receptor antagonist, abolished or significantly decreased the evoked responses. These results indicate that excitatory amino acids are the neurotransmitter at the first relay in the taste pathway in the central nervous system.

Parallel medullary gustatospinal pathways in a catfish: possible neural substrates for taste-mediated food search.

Taste and tactile fibers in the facial nerve of catfish innervate extraoral taste buds and terminate somatotopically in the facial lobe (FL)-a medullary structure crucial for gustatory-mediated food search. The present study was performed to determine the neural linkages between the gustatory input and the spinal motor output. Spinal injections of horseradish peroxidase (HRP) label spinopetal cells in the octaval nuclei, the nucleus of the medial longitudinal fasciculus, and reticulospinal neurons (Rsps) in the brainstem medial reticular formation (RF), including the Mauthner cell. A somatotopically organized, direct faciospinal system originating from superficial cells scattered in the lateral lobule of the facial lobe (ll) is also labeled. The brainstem reticulospinal cells are segmentally organized into 14 clusters within eight segments of the reticular formation and includes one cluster (RS5) directly ventral to the FL. Injections of HRP or fluorescent tracers into the medial lobule of the FL label a facioreticular projection terminating around the Rsps of RS5. DiI injections into this area of the RF retrogradely label deeply situated bipolar neurons, especially in the medial and intermediate lobules of the FL. Electrophysiological recordings in and around RS5 show units with large receptive fields and with responses to chemical and tactile stimulation. The FL projects to the spinal cord via two pathways: (1) a topographically organized direct faciospinal pathway, and (2) an indirect facioreticulospinal pathway in which reticular neurons process and integrate gustatory information before influencing spinal circuitry for motor control during food search.

Evolution of taste and solitary chemoreceptor cell systems.

Vertebrates possess four distinct chemosensory systems distinguishable on the basis of structure, innervation and utilization: olfaction, taste, solitary chemoreceptor cells (SCC) and the common chemical sense (free nerve endings). Of these, taste and the SCC sense rely on secondary receptor cells situated in the epidermis and synapsing on sensory nerve fibers innervating them near their base. The SCC sense occurs in anamniote aquatic craniates, including hagfish, and may be used for feeding or predator avoidance. The sense of taste occurs only in vertebrates and is always utilized for feeding. The SCC system achieves a high degree of specialization in two teleosts: sea robins (Prionotus) and rocklings (Ciliata). In sea robins, SCCs are abundant on the three anterior fin rays of the pectoral fin which are free of fin webbing and are used in active exploration of the substrate. Behavioral and physiological studies show that this SCC system responds to feeding cues and drives feeding behavior. It is connected centrally like a somatosensory system. In contrast, the specialized SCC system of rocklings occurs on the anterior dorsal fin which actively samples the surrounding water. This system responds to mucus substances and may serve as a predator detector. The SCC system in rocklings is connected centrally like a gustatory system. Taste buds contain multiple receptor cell types, including a serotonergic Merkel-like cell. Taste receptor cells respond to nutritionally relevant substances. Due to similarities between SCCs and one type of taste receptor cell, the suggestion is made that taste buds may be compound sensory organs that include some cells related to SCCs and others related to cutaneous Merkel cells. The lack of taste buds in hagfish and their presence in all vertebrates may indicate that the phylogenetic development of taste buds coincided with the elaboration of head structures at the craniate-vertebrate transition.

Feeding patterns and brain evolution in ostariophysean fishes.

The sense of taste plays a crucial role in a fish's ability to locate and select appropriate food. Functionally, the taste system is divisible into two subsystems, with external taste, utilized to locate food in the environment, being mediated by the facial nerve while intraoral taste, crucial for triggering swallowing, is mediated by the vagus nerve. Each of these nerves connects to its own portion of the medullary viscerosensory column. In most teleosts, the viscerosensory column forms a continuous, relatively undifferentiated column of neuropil in the dorsomedial medulla. The taste bud-bearing surfaces of the fish are mapped onto this column with external taste buds being represented anteriorly and pharyngeal taste buds caudally. Taste information reaching the vagal taste area, the "vagal lobe", is relayed directly to motoneurons that control the oropharyngeal musculature. In goldfish, unlike most teleosts, the vagal lobe is laminated, highly differentiated structure containing both sensory and motor layers. This derived neural structure is related to the specialized palatal food sorting apparatus utilized by the fish to separate food from substrate material. Despite the complex morphology of the vagal lobe in goldfish, the underlying circuitry is essentially identical to that of other fishes, i.e. after an obligatory synapse in the sensory layers, the gustatory input is relayed to the oropharyngeal motoneurons comprising the motor layer. Thus evolution of the derived, laminated brain structure did not entail generation of new connectivity but merely involved rearrangement of previously existing neuronal populations.

Differential localization of putative amino acid receptors in taste buds of the channel catfish, Ictalurus punctatus.

The taste system of catfish, having distinct taste receptor sites for L-alanine and L-arginine, is highly sensitive to amino acids. A previously described monoclonal antibody (G-10), which inhibits L-alanine binding to a partial membrane fraction (P2) derived from catfish (Ictalurus punctatus) taste epithelium, was found in Western blots to recognize a single band, at apparent MW of 113,000 D. This MW differs from the apparent MW for the presumed arginine receptor identified previously by PHA-E lectin affinity. In order to test whether PHA-E lectin actually reacts with the arginine-receptor, reconstituted membrane proteins partially purified by PHA-E affinity were used in artificial lipid bilayers. These reconstituted channels exhibited L-arginine-activated activity similar to that found in taste cell membranes. Accordingly, we utilized the PHA-E lectin and G-10 antibody as probes to differentially localize the L-alanine and L-arginine binding sites on the apical surface of catfish taste buds. Each probe labels numerous, small (0.5-1.0 micron) patches within the taste pore of each taste bud. This observation suggests that each bud is not tuned to a single taste substance, but contains putative receptor sites for both L-arginine and L-alanine. Further, analysis of double-labeled tissue reveals that the PHA-E and G-10 sites tend to be separate within each taste pore. These findings imply that in catfish, individual taste cells preferentially express receptors to either L-arginine or L-alanine. In addition, PHA-E binds to the apices of solitary chemoreceptor cells in the epithelium, indicating that this independent chemoreceptor system may utilize some receptor sites similar to those in taste buds.