Proc. Natl Acad. Sci. USA Vol.'79, pp. 6742-6746, November 1982 Neurobiology Localization of immunoreactive dynorphin in neurons cultured from spinal cord and dorsal root ganglia (endorphin/peptidergic neurons/spinal sensory neurons/immunohistochemistry/radioimmunoassay) PAUL M. SWEETNAM*, JOSEPH H. NEALE*tt, JEFFERY L. BARKERt, AND AVRAM GOLDSTEIN§ *Department of Biology, Georgetown University, Washington, D.C. 20057; tLaboratory of Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205; and §Addiction Research Foundation and Department of Pharmacology, Stanford University, Palo Alto, California 94304 Contributed by Avram Goldstein, August 12, 1982 ABSTRACT Antisera specific for dynorphin were used to with [Leu]enkephalin (9). Low levels ofir-dynorphin also have study the cellular distribution ofopioid peptides in spinal cord and been observed in adult rabbit and rat dorsal root ganglia (11) dorsal root ganglion neurons in dissociated cell culture. Radioim- with the "Lucia" antiserum, whereas ir-enkephalin has been munoassay of 4-wk-old cultures yielded levels of dynorphin im- reported in spinal ganglia that were maintained in explant cul- munoreactivity similar to those in adult rodent spinal cord. Im- ture with spinal cord tissue (12). munohistochemistry showed staining confined to the perinuclear In the present study, we used the "Lucia" antiserum and region of neuronal cell bodies. In contrast, enkephalin immuno- affinity-purified dynorphin antibodies from a very similar anti- reactivity was found in extensive neurite fields as well as in neu- serum ("Paul") to study the cellular distribution ofir-dynorphin ronal perikarya. Opioid peptide immunoreactivity was observed within spinal cord and sensory neurons in dissociated cell cul- in =4% of the spinal cord neurons either with dynorphin or en- tures, and we have compared the results with those obtained kephalin antiserum. No substantial increase in the number of re- antisera. active cells was observed when the two sera were, applied simul- using enkephalin taneously. These results suggest that the perinuclear region of opioid spinal cord neurons in culture contains peptide with an MATERIALS AND METHODS amino acid sequence similar to that of the midportion of dynor- phin, whereas the neurites appear to contain smaller peptides re- Preparation ofCell Cultures. Spinal cord cell cultures were lated to NH2-terminal fragments of dynorphin. By using simple prepared and maintained as described (13). Spinal cords with morphological criteria, spinal sensory neurons can be identified associated sensory ganglia were removed from 13.5- to 14.5-day in these cell cultures and in cultures prepared from dorsal root (gestational age) fetal mice (C57BL/6), mechanically disso- ganglia without spinal cord. Approximately 1-2% ofthese ganglion ciated, and sterilely inoculated in Eagle's minimal essential cells showed intense immunostaining with an affinity-purified dy- medium with 10% horse serum and 10% fetal calf serum. For norphin antiserum. An additional few percent ofthe sensory neu- fluorescence IHC, one-third of a spinal cord was inoculated rons showed less intense opioid immunoreactivity. This result ex- onto collagen-coated 22 x 22mm glass coverslips (Clay Adams). tends the observations of opioid peptides one step further along For peroxidase-antiperoxidase assays, cells from one-third of a the pathway that processes sensory information. spinal cord were inoculated onto collagen-coated 35-mm plastic culture dishes, and for RIA, the cells were inoculated onto 60- Considerable evidence has been obtained to support the in- mm plastic culture dishes at a density ofthree spinal cords per volvement ofopioid peptides in spinal cord function (1). Spinal dish. Background cell growth was inhibited after one treatment cord tissue contains a relatively high concentration of opiate with uridine at 50 pZg/ml and fluorouridine deoxyribose at 20 receptors (2) and enkephalin-like molecules, as defined by im- ,ug/ml (courtesy of W. E. Scott, Hoffinann-LaRoche) applied munohistochemistry (IHC; refs. 3 and 4). Using spinal cord cell between day 4 and day 7 in culture. cultures as a model for the study of these peptides, we have Cells were maintained between day 4 and day 28 in Eagle's reported multiple, distinctly different actions ofopioid peptides minimal essential medium with 10% horse serum. The cultures on the excitability of spinal cord neurons (5, 6) and have dem- that were prepared for RIA determination of dynorphin were onstrated the presence of immunoreactive (ir)-opioid peptide maintained for 4 wk in medium with sodium bicarbonate at 3.5 in cell bodies and neurites, using enkephalin antisera (7). Fur- g/liter and an atmosphere of7% CO2 in air. Cells prepared for ther, we have demonstrated (8) that cultured spinal-neurons IHC were grown either in the above medium or were main- synthesize [Met]enkephalin in a ribosome-dependent manner tained in an air atmosphere with similar medium containing and that the newly synthesized peptide can be released by el- sodium bicarbonate at 0.35 g/liter and 30 mM Hepes buffer evating the extracellular potassium concentration. However, (pH 7.35). We have found the Hepes-buffered medium to pro- beyond this latter study, very little is known about the precise vide a suitable environment for the differentiation of murine structure of the opioid peptides within spinal cord neurons. spinal cord and brain cells in dissociated culture. There was no Highly specific antiserum, "Lucia" (9), prepared against the apparent difference in the expression ofpeptide development first 13 amino acids of the 17-residue dynorphin, has revealed between cultures maintained in either buffer system. that spinal cord contains a rather high concentration of this IHC. Cultures were rinsed with medium and fixed for 40 min opioid peptide (10, 11). This antiserum has been characterized in freshly depolymerized, cold, 4% paraformaldehyde in 0.1 M by radioimmunoassay (RIA) as exhibiting maximal affinity for phosphate buffer. Rabbit antiserum to dynorphin ("Lucia" or the sequence 3-12 of dynorphin, with < 10-6% crossreactivity "Paul"), [Metlenkephalin (lot no. 49279, ImmunoNuclear), or The publication costs ofthis article were defrayed in part by page charge Abbreviations: RIA, radioimmunoassay; IHC, immunohistochemistry; payment. This article must therefore be hereby marked "advertise- ir, immunoreactive. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 6742 Downloaded by guest on September 25, 2021 Neurobiology: Sweetnam et d Proc. Natd Acad. Sci. USA 79 (1982) 6743 [Leu]enkephalin (generously provided by June L. Dahl, De- antibodies were obtained by immunoaffinity purification on partment of Pharmacology, University of Wisconsin Medical Sepharose 4B coupled to dynorphin-(1-13). In RIA, crossreac- School) was adsorbed with mouse liver powder at 20 gg/ml tivities of this antibody preparation against dynorphin-(1-13) (Cappel Laboratories, Cochranville, PA) to decrease nonspe- standard were: dynorphin-17, 100%; a-neo-endorphin, 8 X cific reactivity in the IHC. 10-5%; dynorphin B (16), 7 X 10-5%; [Leu]enkephalin, 3 X For fluorescence studies, the fixed cultures were incubated 10-6%. Specificity of"Lucia" in IHC, determined by blocking with 0.5 ml of a 1:100 dilution of rabbit antiserum with phos- experiments, is reported in Results and Discussion. phate-buffered saline (pH 7.4) for up to 24 hr in a humid en- vironment at 40C. By using the indirect immunofluorescence method of Coons (14), the cultures were incubated for 30 min RESULTS AND DISCUSSION at 40C with a 1:40 dilution with phosphate-buffered saline (pH Cells dissociated from embryonic mouse spinal cord and sensory 7.4) offluorescein-conjugated goat antiserum against rabbit IgG ganglia contained ir-dynorphin after 4 wk of differentiation in (Cappel Laboratories). Cultures were inverted on a glass slide culture (Table 1). This level ofpeptide is equivalent to that re- with a drop ofglycerol and phosphate-buffered saline, 3:1 (vol/ ported in the dorsal spinal cord ofadult rodents (11). The data vol), and visualized in a Zeiss Photoscope. For the enzyme- are consistent with the results obtained after spinal cord tran- mediated histochemistry (15), fixed cells were incubated as fol- section, which indicated that most of this peptide was present lows: 1 hr at room temperature with 3% normal goat serum in in the spinal neurons rather than in descending spinal afferents 50 mM Tris HCI-buffered (pH 7.4) saline; 24 hr at 40C or 3 hr (11). The RIA employed in this study was quite specific for the at room temperature in a 1:600 dilution of primary dynorphin midportion amino acid sequence in dynorphin. The lack ofsig- or enkephalin antiserum with 0.25% Triton X-100/1% normal nificant crossreactivity with the enkephalins in this assay to- goat serum/50 mM Tris-HCI-buffered saline; 30 min at room gether with our previous demonstration-by using HPLC-of temperature in a 1:40 dilution of goat anti-rabbit IgG (Stern- [Met]enkephalin synthesis and release by similar spinal cord berger-Meyer Immunochemicals); 30 min at room temperature cell. cultures clearly indicate the presence of two different with a 1:80 dilution of the peroxidase-antiperoxidase complex opioid peptides in spinal cord neurons. (Sternberger-Meyer Immunochemicals); and finally, 6-20 min Using RLA we were unable to demonstrate release of a sig- at 220C with 0.06% 3,3'-diaminobenzidine tetrahydrochloride/ nificant amount of dynorphin from these cultures. However, 0.01% hydrogen peroxide in Tris buffer. this negative result may not be conclusive for several reasons. RIA. Spinal cord cells in 60-mm culture dishes were rinsed We might anticipate release of<5% oftotal neuronal dynorphin and incubated in 2.5 ml of Earle's balanced salt solution con- under the conditions of elevated extracellular potassium. This taining 0.5% bovine serum albumin, bacitracin at 150 mg/li- would represent a level ofpeptide close to the assay background ter, and glucose at 4.5 g/liter for 30 min at 37C.