BIGIANI AND ROPER Cell Types in Necturus Taste Buds 165 Electrophysiological Subtypes : Basal Cells

We used the patch clamp technique to record from taste cells in thin transverse slices of lingual epithelium from Necturus maculosus. In this preparat ion, the epithelial polarity and the cellular organization of the taste buds, as well as the interrelationships among cells within the taste bud, were preserved. Whole-cell recording, combined with cell identification using Lucifer yellow, allowed us to identify distinct subpopulations of taste cells based on their electrophysiological properties. Receptor cells could be divided in two groups: one group was characterized by the presence of voltage-gated Na +, K +, and Ca 2+ currents; the other group was characterized by the presence of K + currents only. Therefore, receptor cells in the first group would be expected to be capable of generating action potentials, whereas receptor cells in the second group would not. Basal taste cells could also be divided into two different groups. Some basal cells possessed voltage-gated Na +, K +, and Ca 2+ conductances, whereas other basal cells only had K + conductance. In addit ion to single taste cells, we were able to identify electrically coupled taste cells. We monitored cell--cell coupling by measuring membrane capacitance and by observing Lucifer yellow dye coupling. Electrical coupling in pairs of dye-coupled taste receptor cells was strong, as indicated by experiments with the uncoupling agent 1-octanol. I~lectrically coupled receptor cells possessed voltage-gated currents, including Na + and K + currents. The electrophysiological differentiation among taste cells presumably is related to functional diversifications, such as different chemosensitivities. I N T R O D U C T I O N Tas te r ecep to r cells have been shown to be coup led via chemical and electrical synapses, rais ing the possibili ty that some degree o f signal process ing occurs in taste buds (Roper , 1992). Very little is known about the extent and funct ional significance of synaptic interact ions in taste buds, especially electrical coupl ing. West and Berna rd (1978) indica ted that some cells in taste buds were electrically and dye coupled. Subsequent studies have e x t e n d e d and ref ined these initial repor ts . I t is now well Address correspondence to Dr. S. D. Roper, Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523. j . GEN. PHYSIOL. © The Rockefeller University Press • 0022-1295/93/07/0143/28 $2.00 Volume 102 July 1993 143-170 143 on D ecem er 2, 2017 jgp.rress.org D ow nladed fom 144 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 102 • 1993 established that subsets of taste receptor cells are coupled in groups of approximately two or three when examined with Lucifer yellow (Teeter, 1985; Yang and Roper, 1987; Sata and Sato, 1989). The physiological properties of these cells and how these properties compare with those from adjacent, noncoupled taste cells have not been elucidated. This report is the first in a series on the functional characteristics of electrically coupled and noncoupled taste cells in a new, semi-intact preparation of lingual epithelium from Necturus maculosus. Taste bud cells are large in this species (Farbman and Yonkers, 197 l) and thus are amenable to detailed electrophysiological investigations, including patch recording. To date, patch clamp recording has been used to study the electrical properties of isolated taste cells in Necturus, as well as other species (Ambystoma: Sugimoto and Teeter, 1990; catfish: Teeter, Brand, and Kumazawa, 1990; frog: Avenet and Lindemann, 1987a, 1988; Avenet, Hofmann, and Lindeman, 1988; Miyamoto, Okada, and Sato, 1988; mouse: Spielman, Mody, Brand, Whitney, MacDonald, and Saher, 1989; Necturus: Kinnamon, Cummings, and Roper, 1988a; Kinnarnon, Dionne, and Beam, 1988b; Kinnamon and Roper, 1988a; Cummings and Kinnamon, 1992; rat: Akabas, Dodd, and A1-Awqati, 1988, 1990; Herness, 1989; Behe, DeSimone, Averlet, and Lindemann, 1990). Recently, however, it has been shown that patch clamp recordings can be successfully obtained in thin slices of nervous tissue without the use of proteolytic enzymes and cell isolation (Blanton, Lo Turco, and Kriegstein, 1989; Edwards, Konnerth, Sakmann, and Takahashi, 1989; reviewed in Konnerth, 1990). This has introduced the possibility of using patch recording of taste buds in slices of lingual epithelium to study synaptic connections, particularly electrical synapses, with voltage clamp techniques. Here we show that a slice preparation of Necturus lingual epithelium, used previously for intracelfular recording studies (Bigiani and Roper, 1991a; Ewald and Roper, 1992a), is suitable for patch recording techniques without any enzymatic treatment. In this preparation the epithelial polarity and the cellular organization of the taste bud, as well as the interrelationships among cells within the taste bud, are preserved. Therefore, taste cells can be studied electrophysiologically in a cellular environment that more closely resembles that in vivo. We have identified coupled cells and monitored electrical coupling with a single patch recording electrode by measuring membrane capacitance (e.g., Santos-Sacchi, 1991). This report focuses on the voltage-dependent ionic currents in electrically coupled and noncoupled taste cells in Necturus in the lingual slice preparation. The findings show that there are distinct subpopulations of cells within the taste bud that can be distinguished on the basis of their electrophysiological properties. Preliminary reports of part of this work have been presented in abstract form (Bigiani and Roper, 1991b, c; Bigiani, Avenet, and Roper, 1992). MATERIALS AND METH O D S Tissue Preparation and Optical Setup Mudpuppies (Necturus maculosus) werc obtained irom commercial suppliers and maintained at 4-10°C in fresh water aquaria. They were fed minnows weekly. The procedure to obtain lingual slices has been previously described (Bigiani and Roper, on D ecem er 2, 2017 jgp.rress.org D ow nladed fom BIO1ANI AND ROPER Cell Types in Necturus Taste Buds 145 1991a; Ewald and Roper, 1992a). Briefly, mudpuppies were anesthetized in ice water and rapidly decapitated. The tongue was removed and placed in cold (4-5°C) amphibian physiological solution (APS; see below). The lingual epithelium was gently freed by blunt dissection. The lingual tissue, with the epithelial surface oriented upward, was carefully fixed to a block of firm supporting material, such as carrot, with cyanoacrylic glue. Transverse slices (~ 200 ~m thick) of the lingual epithelium were cut with a vibrating razor blade (a modified ladies' Remington electric shaver mounted on a micromanipulator, as suggested to us by A. R. Cinelli and J. S. Kauer) while the tissue was kept moist with ice-chilled APS. Slices of lingual tissue were scanned with a compound microscope at 50x. Sections containing taste buds were selected and pinned out in a shallow recording chamber ( ~ 1 ml vol) containing APS. The chamber was placed onto the stage of a fixed-stage upright Zeiss microscope equipped with a 40x water immersion objective (Nikon CF, working distance = 2.0 mm). When viewed this way (i.e., at 500x), entire taste buds with their cellular organization were clearly visible in the preparation (Fig. 1 A ). During the experiments, the tissue was continuously perfused with APS (flow rate: ~ 3-5 ml/min) by means of a gravity-driven system.