Intracellular dye-marked enkephalin neurons in the magnocellular preoptic nucleus of the goldfish hypothalamus.

Abstract

A method that combines intracellular recording, dye marking, and immunocytochemistry makes the study of functional and morphological aspects of enkephalin neurons in the magnocellular preoptic nucleus of the goldfish hypothalamus feasible. By use of multiple techniques, enkephalin neurons can be distinguished from other brain cells and can be reconstructed from drawings of serial sections containing the dye-injected opioid cells. These enkephalin cells and their processes measure 14-42,um provided important data on neuronal responses to discrete physical, chemical, and behavioral stimuli (1). Despite these maneuvers, investigators have been unable to identify or directly classify these functionally studied cells chemically. The recent discovery of enkephalin in the magnocellular nuclei, the hypothalamus, and the pituitary gland of the goldfish (2-5) confirms earlier studies in mammals (6-10) and suggests a role for endogenous opioid peptides in neurohypophyseal regulation in fish and mammals. The combination of cell identification by immunocytochemistry with a previously developed intracellular dye-injecting approach in the goldfish (11) provides a working model for chemical typing of single cells. This investigation offers an approach to the analysis of neural networks by use of the current technology of cell identification by dye marking (12), peptide biochemistry (13), and immunocytochemistry (6) for the study of single, electrophysiologically defined, morphologically reconstructed magnocellular neurons that contain enkephalin. MATERIALS AND METHODSAdult goldfish (Carassius auratus), weighing 290-850 g and measuring 25-35 cm, were anesthetized, immobilized, suspended, and perfused with water, and the brain was exposed according to earlier methods (11,14).Glass micropipettes were filled with a 3% aqueous solution of Lucifer Yellow-CH (LY; see ref. 12) and beveled (15) to impedances of 6-40 MQ for intracellular recordings. Electrophysiological recording and stimulating apparatus and the pituitary stimulating electrodes have been described (11). Intracellular electrophoretic injection (5-10 nA of hyperpolarizing dc current, 0.3-6 min) of LY (an anionic fluorescent dye) marked the physiologically studied, antidromically identified preoptic neurons for subsequent localization. In most fish, only one cell per nucleus was filled with LY. At the conclusion of each experiment, the fish was perfused with teleost physiological solution (16) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Photoconversion of diaminobenzidine with different fluorescent neuronal markers into a light and electron microscopic dense reaction product

“…Its popularity is due to its intense fluorescence, stability during illumination, and compatibility with a variety of embedding procedures. As a result, LY is now used in a variety of biological systems (Buhl and Dann, 1988;Buhl and Lubke, 1989;Buhl and Schlote, 1987;Buhl et al, , 1990Einstein, 1988;Katz, 1987;Lubke and Albus, 1989;Reaves and Hayward, 1979;Rho and Swanson, 1987;Smithson et al, 1983;Masland, 1984, 1985;Wouterlood et al, 1990; see also Djamgoz and Kolb, this issue; Meredith and Wouterlood, this issue). After the introduction in fixed tissue (LY injection into retinal ganglion cells; Tauchi and Masland, 19841, this technique in area 17 of a 5-day-old kitten.…”

“…In addition to older neuroanatomical methods such as the classical Golgi-impregnation technique, degeneration techniques, and other methods based on the transport of large macromolecliles, tracing with fluorescent dyes and intracellular injections with LY are relatively new techniques in neurobiology. The latter fluorescent dyes were introduced as alternative and additional neuroanatomical markers to study the connectivity and morphology of neurons in the CNS (Bentivoglio and Su, 1990;Buhl and Lubke, 1989;Einstein, 1988;Godeinent et al, 1987;Honig and Hume, 1989;Katz, 1987;Kuypers et al, 1977Kuypers et al, , 1979Lubke and Albus, 1989;Reaves and Hayward, 1979;Sandell and Masland, 1988;Smithson et al, 1983;Stewart, 1978Stewart, , 1981Tauchi and Masland, 1985;Wouterlood et al, 1990). Because DAB can now be photoconverted with a number of fluorescent markers into a stable reaction product, using a simple protocol originally described by Maranto (1982), the capability of these methods is now no longer restricted to the fluorescence microscope level.…”

Electrophysiology and enkephalin immunoreactivity of identified myenteric plexus neurones of guinea‐pig small intestine.

“…In experiments where Lucifer Yellow was not used the second antibody was that coupled to FITC. When cells had been filled with Lucifer Yellow, a second antibody coupled to rhodamine was used so that the immunofluorescence could be easily distinguished from that of Lucifer Yellow (Reaves & Hayward, 1979). The effect of the immunohistochemical procedures on the Lucifer Yellow fluorescence was monitored at each step and it was found that, although the intensity was reduced, well-filled cells and their processes remained readily identifiable.…”

Synaptic inputs to immunohistochemically identified neurones in the submucous plexus of the guinea‐pig small intestine.

“…After fixation the preparation was dehydrated and rehydrated before being exposed for at least 12 h to an anti-VIP antibody (code 7913 from Dr J. H. Walsh) raised in a rabbit; it was then washed and exposed for 1 h to a second (anti-rabbit) antibody coupled to rhodamine (Cappel Laboratories). Use of rhodamine allowed the immunofluorescence to be distinguished from the Lucifer Yellow fluorescence in the filled cells (Reaves & Hayward, 1979).…”