The Chemical Sensitivity and Electrical Activity of Individual Olfactory Sensory Neurons to a Range of Sex Pheromones and Food Odors in the Goldfish

Abstract

Although it is well established that the olfactory epithelium of teleost fish detects at least 6 classes of biologically relevant odorants using 5 types of olfactory sensory neurons (OSNs), little is understood about the specificity of individual OSNs and thus how they encode identity of natural odors. In this study, we used in vivo extracellular single-unit recording to examine the odor responsiveness and physiological characteristics of 109 individual OSNs in mature male goldfish to a broad range of biological odorants including feeding stimuli (amino acids, polyamines, nucleotides), sex pheromones (sex steroids, prostaglandins [PGs]), and a putative social cue (bile acids). Sixty-one OSNs were chemosensitive, with over half of these (36) responding to amino acids, 7 to polyamines, 7 to nucleotides, 5 to bile acids, 9 to PGs, and 7 to sex steroids. Approximately a quarter of the amino acid-sensitive units also responded to polyamines or nucleotides. Three of 6 amino acid-sensitive units responded to more than 1 amino acid compound, and 5 sex pheromone-sensitive units detected just 1 sex pheromone. While pheromone-sensitive OSNs also responded to the adenylyl cyclase activator, forskolin, amino acid-sensitive OSNs responded to either forskolin or a phospholipase C activator, imipramine. Most OSNs responded to odorants and activators with excitation. Our results suggest that pheromone information is encoded by OSNs specifically tuned to single sex pheromones and employ adenylyl cyclase, suggestive of a labeled-line organization, while food information is encoded by a combination of OSNs that use both adenylyl cyclase and phospholipase C and are often less specifically tuned.

Figure 1.

Electrical characteristics of representative single goldfish OSNs. (A) A high-K-induced single-unit response of an OSN. The bottom bar indicates 5-s application of 30 mM high-K solution. (B) Waveforms of each spike event extracted from a trace shown in (A). The isolation threshold is indicated by a dotted line in (A). A bar indicates 2 ms. (C) Scatter plot illustrating the spike amplitudes (y-axis) and the spike duration at the 50% level of maximal spike amplitude (x-axis). In (B) and (C), pseudocolors indicate the firing rate, with blue and red corresponding to 3 and 30 Hz, respectively. (D) Typical excitatory (top) or inhibitory (bottom) single-unit responses. Each of the top traces in each set of the 3 describes the EOG, while the middle traces describe single-unit activity, and the bottom traces describe the firing rate at each spike event. The spike activity of the top single-unit was excited by the 10^–9 M sex steroid mixture; the other was suppressed by the 10^–4 M amino acid mixture. The bottom bar indicates the timing of 5-s odor stimulation. A vertical bar indicates voltage amplitude in EOG responses. Asterisks denote significant increases in firing rate (P < 0.05). Arrow indicate the significant decreases in firing rate (P < 0.05).

Figure 2.

Response profiles of the 61 chemosensitive single-units tested with all 6 odorant classes (mixtures) (48 insensitive units not shown). The Response Index (RI, relative change in response rate) of each olfactory response is shown by box drawings.

Figure 3.

An example of responses of a single goldfish OSN that responded to both the nucleotide and the amino acid mixture (unit #34 in Figure 2) and no other stimuli other than forskolin. Traces show EOG recordings (top traces of each pair of traces) and single-unit activity (bottom traces) elicited by the nucleotide, amino acid, polyamine, bile acid, prostaglandin, sex steroid mixtures, and 10^–5 M forskolin. The mixture concentration of nucleotide and amino acid is indicated to the right. The vertical bar indicates amplitude of EOG amplitude. The bar at the bottom indicates the 5-s odorant stimulation period. Asterisks denote a significant increase in firing rate (P < 0.05).

Figure 4.

Action potentials of 5 sex pheromone-sensitive single-units to single compounds. In each pair of traces, the top traces indicate EOGs, while the bottom traces indicate single-unit activity, respectively. Bottom bars indicate the timing of odorant stimulation for 5-s. A vertical bar indicates voltage amplitude in EOG responses. #46, #47, and #54 responded specifically to PGF, 15kPGF, 17,20βP-s, respectively. Both #55 and #56 was AD-specific single-units. Asterisks denote significant increase of firing rate (P < 0.05).

Figure 5.

Response profiles of the 30 chemosensitive single-units, which were tested with 10^–5 M forskolin, 10^–4 M imipramine,10^–4 M amino acid mixture, 10^–8 M prostaglandin mixture, and 10^–9 M sex steroid mixture. The RIs of each olfactory response and insensitiveness (no response) are shown as noted by box drawings.

Figure 6.

Four examples of the electrophysiological activity of single goldfish OSNs to pharmacological agents. (A) Activation of activity by exposure to 10^–5 M forskolin in an amino acid-sensitive single-unit (#9 in Figure 5). (B) Activation of activity by 10^–4 M imipramine in an amino acid-sensitive single-unit (#27 in Figure 5). (C) Activation of activity by 10^–5 M forskolin in a sex steroid-sensitive single-unit (#23 in Figure 5). (D) Modulation of the spike activity by 10^–4 M imipramine in a prostaglandin and forskolin-sensitive single units (#24 in Figure 5). In each pair of traces, the top trace shows the EOG while bottom trace indicates single-unit activity. Bottom bars indicate the timing of 5-s odorant stimulation. Asterisks denote significant increase of firing rate (P < 0.05). Arrows indicate a decrease in firing rate (P < 0.05).