John Caprio

Correlation between Olfactory Receptor Cell Type and Function in the Channel Catfish

The olfactory epithelium of fish contains three intermingled types of olfactory receptor neurons (ORNs): ciliated, microvillous, and crypt. The present experiments were undertaken to test whether the different types of ORNs respond to different classes of odorants via different families of receptor molecules and G-proteins corresponding to the morphology of the ORN. In catfish, ciliated ORNs express OR-type receptors and Gαolf. Microvillous ORNs are heterogeneous, with many expressing Gαq/11, whereas crypt ORNs express Gαo. Retrograde tracing experiments show that ciliated ORNs project predominantly to regions of the olfactory bulb (OB) that respond to bile salts (medial) and amino acids (ventral) (Nikonov and Caprio, 2001). In contrast, microvillous ORNs project almost entirely to the dorsal surface of the OB, where responses to nucleotides (posterior OB) and amino acids (anterior OB) predominate. These anatomical findings are consistent with our pharmacological results showing that forskolin (which interferes with Gαolf/cAMP signaling) blocks responses to bile salts and markedly reduces responses to amino acids. Conversely, U-73122 and U-73343 (which interfere with Gαq/11/phospholipase C signaling) diminish amino acid responses but leave bile salt and nucleotide responses essentially unchanged. In summary, our results indicate that bile salt odorants are detected predominantly by ciliated ORNs relying on the Gαolf/cAMP transduction cascade. Nucleotides are detected by microvillous ORNs using neither Gαolf/cAMP nor Gαq/11/PLC cascades. Finally, amino acid odorants activate both ciliated and microvillous ORNs but via different transduction pathways in the two types of cells.

Polyamines as olfactory stimuli in the goldfish Carassius auratus.

SUMMARY Electrophysiological responses of goldfish olfactory receptor neurons (ORNs) and goldfish behavioral responses to polyamines were investigated in vivo. Electro-olfactogram (EOG) recordings indicated that polyamines (putrescine, cadaverine and spermine) are potent olfactory stimuli for goldfish with estimated electrophysiological thresholds of 10–8–10–7 mol l–1, similar to that for L-arginine, the most stimulatory amino acid. Although thresholds were similar, the magnitude of the EOG responses to intermediate (10–5–10–4 mol l–1) and high (10–3 mol l–1) concentrations of polyamines dwarfed the responses to amino acids and related single amine containing compounds (amylamine and butylamine). The EOG responses to 0.1 mmol l–1 putrescine, cadaverine and spermine were, respectively, 4.2×, 4.3× and 10.3× the response of the standard, 0.1 mmol l–1 L-arginine. Electrophysiological cross-adaptation experiments indicated that polyamine receptor sites are independent from those to L-amino acids (alanine, arginine, glutamate, lysine, methionine and ornithine), bile salts (sodium taurocholate and taurolithocholate), the single amine containing compounds (amylamine and butylamine) and ATP. Further, the cross-adaptation experiments revealed the existence of independent receptor sites for the different polyamines tested. Pharmacological experiments suggested that polyamine odorant transduction does not primarily involve the cyclic AMP and IP3 second messenger pathways. Behavioral assays indicated that polyamines are attractants that elicit feeding behavior similar to that elicited by L-amino acids.

Taste and tactile recordings from the ramus recurrens facialis innervating flank taste buds in the catfish

Summary1.The ramus recurrens facialis (VII) innervating the flank of the channel catfish,Ictalurus punctatus, responded to both amino acid (Fig. 2–10; Table 1) and mechanical (Fig. 11) stimulation of the flank skin.2.The integrated phasic gustatory responses generally increased exponentially with logarithmic increase in stimulus concentration over a wide concentration range with a tendency for saturation at concentrations ≧10−4.5 M (Fig. 6).3.l-alanine andl-arginine HCl were the most stimulatory compounds tested, with thresholds averaging 10−8.6±0.4m (M±SE) (Fig. 4; Table 1).4.Cross-adaptation experiments suggest that alanine and arginine bind to relatively independent receptor sites on the taste cells (Fig. 10; Table 1); further, single-unit studies indicate the recurrent nerve is composed of at least two types of amino acid sensitive taste fibers, alanine-best and arginine-best fibers (Fig. 8). In general, unit responses were consistent with the multiunit data (Table 1).5.Taste fibers of the recurrent nerve are relatively insensitive to sodium chloride and sucrose; generally, responses were obtained to quinine hydrochloride and hydrochloric acid, but only at relatively high stimulus concentrations (≧10−4 M, Fig. 7).6.Taste responses of the recurrent nerve are similar to the results obtained from maxillary barbel taste recordings in the same species (Caprio 1975, 1978), suggesting an analogous chemical response profile of facially innervated taste buds irrespective of their anatomical location in the catfish.7.Intra-cranial neural recordings from the recurrent nerve indicate that the tactile responsiveness observed in the present experiments was not attributable to spinal innervation of the flank. The size and contour of the mechanoreceptive flank region innervated by teased bundles of the recurrent nerve varied greatly; some single units responded to both chemical and mechanical stimuli (Fig. 11).

An electrophysiological investigation of the oro-pharyngeal (IX–X) taste system in the channel catfish,Ictalurus punctatus

Summary1.The chemospecificities of the oropharyngeal taste buds innervated by the glossopharyngeal (IX) and vagus (X) nerves were similar; both (IX) and (X) taste systems were highly responsive tol-alanine andl-arginine which have been shown to be the most effective stimuli of the facial (VII) taste system.2.The integrated phasic gustatory responses generally increased exponentially with logarithmic increase in stimulus concentration over a wide concentration range (4 to 6 log units).3.Thresholds forl-alanine andl-arginine averaged (in mol/l) 10−7.4±0.3 (IX) and 10−7.0±0.4 (X) and 10−8.0±0.2 (IX) and 10−6.9±0.4 (X) (mean±S.E.) respectively; for all other amino acids tested thresholds were ≧10−6 mol/l.4.Among the various conventional taste stimuli tested (sodium chloride, sucrose, hydrochloric acid and quinine hydrochloride), quinine was the most stimulatory with thresholds ranging between micromolar and nanomolar concentrations.5.Cross-adaptation experiments suggested the presence of relatively independent receptor mechanisms for at least four of the compounds tested:l-alanine,l-arginine hydrochloride,l-proline and quinine hydrochloride.6.The majority of fiber bundles in both the glossopharyngeal and vagal nerves were highly sensitive to tactile stimulation. Tonically firing proprioceptive units with response frequencies dependent on the position of a gill arch were observed.7.Muscle contractions were observed following the application of high concentrations of certain stimuli (l-arginine and sodium chloride), indicating the chemospecific nature of such reflexes with a possible role in swallowing or rejection of potential food material.8.These data in conjunction with reports on the facial (VII) taste system indicate that similar amino acids may provide important cues for both food searching and ingestive behaviours in the channel catfish.

Peripheral Filters and Chemoreceptor Cells in Fishes

Olfaction in vertebrates is the sense used in the processing of chemical information that is detected and transmitted to the central nervous system by bipolar neurons that compose the olfactory nerve (cranial nerve I). Gustation is the sense used in the processing of chemical information that is detected by taste cells and transmitted centrally by facial (cranial nerve VII), glossopharyngeal (IX), or vagal (X) nerves. Receptor cells of both systems must discriminate relevant chemical stimuli from background chemical “noise” that exists in the immediate environment of all vertebrates. This ability of chemoreceptors to act as “peripheral filters” by detecting and preferentially passing information concerning biologically important chemical stimuli occurs through the evolutionary development of different receptor molecules with different chemospecificities built into the membranes of the receptor cells. This chapter will review the general organization of the olfactory epithelium and taste bud structure in fishes and summarize recent physiological findings as they relate to the sensitivities and specificities of the “filtering elements” (i.e., transduction processes and types of receptor sites) of the receptive olfactory and gustatory receptor cells. For books specific to chemoreception in aquatic organisms, see Kleerekoper (1969), Mackie and Grant (1974), Hara (1982), and Hasler and Scholz (1983).

Ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish, Carassius auratus.

The ultrastructure of the olfactory epithelium in intact, axotomized, and bulbectomized goldfish was studied by scanning and transmission electron microscopy. A total of 58 adult goldfish of various survival times were examined to determine whether the different types of surgery—either olfactory nerve transection or bulbectomy—yielded differences in the extent or time course of cellular degeneration and renewal. Control animals were also examined in detail to elucidate previous controversial findings concerning the types of olfactory receptor neurons present in goldfish. We found that the intact olfactory epithelium of unoperated control goldfish contains the previously observed ciliated and microvillous receptor neurons, and the crypt cell, a cell type not yet seen in the goldfish but recently reported in other species of teleosts. Following either olfactory nerve transection or bulbectomy, the olfactory receptor neurons showed similar signs of degeneration and subsequent cell death, but, surprisingly, the thickness of the olfactory epithelium did not change significantly with either treatment. The time course of receptor cell renewal was different in axotomized and bulbectomized goldfish. In axotomized goldfish, the amount of receptor cells decreased continuously until 8–13 days after surgery, followed by rapid cell renewal. For bulbectomized goldfish, cell replacement began almost immediately after surgery, with degeneration and cell renewal occurring simultaneously. Six weeks after bulbectomy, cell death and cell proliferation reached a “steady state,” and the epithelia did not further improve. Microsc. Res. Tech. 45:325–338, 1999. 

Learned olfactory discrimination versus innate taste responses to amino acids in channel catfish (Ictalurus punctatus).

Intact channel catfish conditioned to the L-amino acids, proline, arginine, alanine, and lysine, discriminated these stimuli from all other amino acids tested. Behavioral structure-activity tests indicated that L-pipecolate was the only effective agonist of the L-proline conditioned response. For channel catfish in which one of the paired olfactory organs was surgically removed, the number of turns to the conditioned stimulus was 40% fewer than those of intact catfish; however, these semiosmic channel catfish discriminated the conditioned from nonconditioned stimuli, as evidenced by their responding to the conditioned amino acid, with a two- to threefold greater number of turns than to the nonconditioned amino acids. Irrespective of the number of conditioning trials attempted, catfish with both olfactory organs removed were unable to discriminate the conditioned from the nonconditioned stimuli.

Electrophysiological evidence for the topographical arrangement of taste and tactile neurons in the facial lobe of the channel catfish

Neural responses in the facial lobe of th channel catfish to chemical and mechanical stimulation of the external skin surface were studied electrophysiologically. Taste and tactile neurons in the lobe were organized in a somatotopic manner, which confirms the anatomical reports of the facial lobe in the bullhead catfish, but is markedly different from that of the Cyprinidae. The taste neurons were arranged generally in the more dorsal regions of the tactile sensitive areas and responded with highest frequency to L-alanine or L-arginine HCI among several amino acids tested. The mechanically responsive neurons in the deeper layer of the antero-medial portion of the lobe, possibly corresponding to the intermediate nucleus of the facial lobe, had large receptive fields ranging from 100 mm to the whole body surface; in addition, some of these neurons showed lateral inhibition. The present study revealed that the facial lobe of the channel catfish is a center not only for gustatory input, but also for tactile information.

Comparison of olfactory receptor (EOG) and bulbar (EEG) responses to amino acids in the catfish,Ictalurus punctatus

The olfactory bulb electroencephalogram (EEG) has been used as a method to imply receptor events12,13. However, experiments to correlate olfactory receptor and bulbar EEG activity in the same species of fish has not been performed. Reported here is the comparison between the simultaneously recorded receptor electroolfactogram (EOG) and the bulbar EEG in the channel catfish, Ictalurus punctatus. With amino acid stimulation of the olfactory mucosa, both the EOG and EEG exhibited an initial phasic response followed by a tonic level maintained throughout the stimulus duration. The relative magnitude of the tonic EEG activity (tonic level/phasic response), however, was significantly less than that for the EOG. Both EOG and integrated EEG responses increased exponentially with logarithmic increase in stimulus concentration from threshold to 10(-3) M. Estimated electrophysiological thresholds for 5 amino acids tested determined by both recording methods did not differ significantly and averaged 10(-9.3) +/- 0.2 M for the EOG and 10(-9.1) +/- 0.2 M for the EEG. There was also a significant correlation between the order of relative effectiveness for 11 amino acids determined by EOG and EEG recordings. These results indicate that in the catfish the olfactory bulb EEG is an indicator of olfactory receptor activity.

Consummatory feeding behavior to amino acids in intact and anosmic channel catfish Ictalurus punctatus

The entire sequence of feeding behavior patterns exhibited by intact and anosmic channel catfish to food extracts was also released by single amino acids. L-arginine (> 10(-6) M), L-alanine (> 10(-6) M), and L-proline (> 10(-4) M) were each highly effective at releasing consummatory behavior patterns, such as turning, increasing pumping of water across the gill arches, and biting-snapping. Swallowing required solid objects, whereas rhythmic movement of the hyoid was released by > 10(-2) M L-arginine alone. For the biting-snapping behavior, the number of bites depended upon both the number of eddies containing the amino acid above the behavioral threshold concentration and the amino acid applied. Multiple eddies of > 10(-3) M L-proline and L-alanine provoked up to 25 bites per test; however, the most effective stimulus for releasing biting-snapping behavior at low concentrations was L-arginine (behavioral threshold 3 x 10(-7) M). In comparison to 10(-4) M L-alanine and L-arginine, other amino acids were less effective stimuli.