Neuroendocrinology 28; 131-137 (1979)

Luteinizing Hormone-Releasing Hormone (LH-RH) Neurons in Cultures of Fetal Rat Hypothalamus1'2 S. C. Feldman3, A. B. Johnson, M.B. Bornstein and G.T. Campbell* Departments of Neurology, Pathology and Neuroscience and the Rose F. Kennedy Center of Research in Mental Retardation and Human Development, Albert Einstein College of Medicine. Bronx. N.Y. and ‘ Department of Physiology and Biophysics, University of Nebraska Medical College, Omaha, Nebr.

Key Words. Hormone ■Neurosecretion • LH-RH-neurons • Hypothalamus • Organotypic culture • Rat fetus

' This work was supported by National Institute of Mental Health Training Grant MH-06418 and by National Institutes of Health grants NS-09649, NS11920, NS-03356, and AM-19170, and by a grant from the Alfred P. Sloan Foundation. 2 Portions of this work were presented at the Ameri­ can Society for Cell Biology, November, 1977. 3 Present address: Department of Anatomy, College of Physicians and Surgeons, Columbia University, 630 West 168th Street. New York. NY 10032.

Accepted after revision: August 25, 1978

The hypothalamus has been suggested as a central nervous system site for both the dif­ ferentiating and feedback effects of gonadal steroid hormones [Dorner and Staudt, 1969; Raisman and Field, 1973). In order to examine this problem more fully, we have utilized an organotypic tissue culture system to charac­ terize the development of the rat hypothala­ mus. Both catecholamine- and LH-RH-con­ taining neurons have been identified in dis­ persed cell cultures derived from neonatal rat hypothalamus [Knigge el al., 1977). These neurons in vitro appear similar to catechol­ amine and LH-RH neurons described in vivo.

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Abstract. Hypothalamic fragments from 21-day-old fetal rats were cultured in Maximow double-coverslip assemblies for I to 2 months. Neurons containing LH-RH were demonstrated immunohistochemically using an antiserum to LH-RH (Dermody; 1:500-1:4,000). LH-RH was demonstrable only in neuronal perikarya (8 13 urn) and in small ( < I mn) round structures nearby, primarily in explants of the median eminence-arcuate nucleus region. Reactive neurons were not found in explants of the preoptic area and could not be demonstrated in fetal hypo­ thalami at the time of explantation. The presence of mature-looking LH-RH-containing neurons in these cultures suggests that this tissue culture system can be used for the study of hypo­ thalamic development.

Feldman/Johnson/Bornstein/Campbell

132

Materials and Methods Hypothalamic fragments for explantation were obtained from 21-day-old fetal rats (CD strain, Charles River, Mass.). The sex of the fetuses was not determined. Fetuses were removed individually from the uterus, and the hypothalamus, including the preoptic area, was dissected free of the brain. Under a dissecting microscope, explants were obtained from the following areas: (I) median eminence-arcuate nucleus region; (2 ) the remainder of the hypothalamus from the optic chiasm to the mammillary bodies, subdivided into 4 sections, rostral to caudal; and (3) the preoptic area. Fragments were kept briefly in culture medium prior to explantation. At the same time, fetal hypothalami were dissected and fixed im­ mediately in Bouin’s solution for immunohistochemistry. Details of the culture method have been pub­ lished [Bornstein, 1973], Fragments (I per covcrslip) were explantcd onto collagen-coated coverslips and maintained in a Maximow double-coverslip assembly. The nutrient medium consisted essentially of 33% human placental serum, 67% Eagle's minimal essen­ tial medium and glucose (to a concentration of 600 mg%). No antibiotics were added to the medium. Explants remained viable for up to 2 months (the longest period studied) as assessed by light microscopy.

Neurons and processes containing LH-RH were visualized, by immunohistochemitry, in sections of paraffin-embedded explants and uncultured hypo­ thalamus and in whole mounts of cultures. All tissue was fixed in Bouin’s solution (4-6 h). Tissue which was to be embedded in paraffin was dehydrated in a series of graded ethanols, and cleared in cedar wood oil (overnight) and xylene, and sectioned serially (5-7 m/

Luteinizing hormone-releasing hormone (LH-RH) neurons in cultures of fetal rat hypothalamus.

Neuroendocrinology 28; 131-137 (1979) Luteinizing Hormone-Releasing Hormone (LH-RH) Neurons in Cultures of Fetal Rat Hypothalamus1'2 S. C. Feldman3,...
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