Intracellular staining of physiologically identified photoreceptor cells and hyperpolarizing interneurons in the teleost pineal organ

The directly photosensory pineal organ of the rainbow trout functions primarily as a luminance detector. Its neutral output reflects the level of ambient illumination in an almost linear fashion over several orders of magnitude. It may thus transmit information about the daily light-dark cycle to central projection targets in the brain, and exert an important control over putative central oscillators. We have studied single neural elements in the explanted pineal organ of the rainbow trout by combining intracellular recording with intracellular injections of either the fluorescent dye Lucifer Yellow CH or the electron dense marker horseradish peroxidase. After physiological characterization, dye was injected, and the pineal organs were processed for fluorescence or electron microscopy. Horseradish peroxidase-injected cells were selected with light microscopy, and were serially sectioned for electron microscopy. By examining the entire series of ultrathin sections of several labeled cells the following results were obtained. (1) Intensity-graded hyperpolarization that was elicited by light stimuli of all wavelengths could be either purely monophasic at all light intensities, or monophasic at low and intermediate light intensities but with an initial peak transient at response saturation. These two types of responses could be demonstrated to emanate from photoreceptor cells. (2) In addition, an interneuron that responded to light stimulation with intensity-graded hyperpolarizations that decreased in amplitude at high light intensities was identified by analysis of serial ultrathin sections. This interneuron was situated in close opposition to a photoreceptor-like element and another interneuron, both of which contained transcellularly transferred horseradish peroxidase. Transcellular transfer of horseradish peroxidase was repeatedly observed, although in the majority of cases only single cells were labeled. Intracellular injection of Lucifer Yellow CH consistently revealed dye-coupling between photoreceptors and between (inter)neurons. The numbers of labeled elements varied between two and eight cells, after intracellular injection of one cell. The present results indicate that the net neural output of the pineal organ is the result of a relatively complicated neural circuitry, encompassing different types of photoreceptors, interneurons and projection neurons. Electrical coupling between photoreceptors, between neurons, and between photoreceptors and neurons may provide spatial signal averaging. The very slow photoreceptor responses to photic stimulation may provide temporal signal averaging. These two averaging mechanisms might together minimize responses to rapid spatial and temporal changes in the ambient illumination, and thus minimize fluctuations in the neural output of the pineal organ that would be irrelevant to the monitoring of the circadian changes in the photic environment