Tissue & Co]l, 1996 28 (4) 411-425 © 1996 Pearson Professional Ltd. Morphological and immunohistochemical properties of primary long-term cultures of adult guinea-pig ventricular cardiomyocytes with peripheral cardiac neurons M. Horackova 1, R. P. Croll 1, D. A. Hopkins 2, A. M. Losier 2, J. A. Armour ~ Abstract. Long-term (2-12 weeks) cultures of adult guinea-pig ventricular myocytes, cocultured with neu- rons derived from stellate or intrinsic cardiac ganglia, retain their functional properties (Horackova et al., 1993, 1994, 1995). The present study was designed to investigate the morphological and immunochemical properties of such neurons and their associated cardiomyocytes. Cultured myocytes studied by means of phalloidin-rhodamine (for F-actin) and an antibody raised against myomes revealed parallel myofibrils with striations typical of rod-shaped cardiomyocytes, even while myo- cytes changed from cylindrical to flattened form as they established intercellular contacts. Microtubular networks, identified by 0~-tubulin DM1A antibody, were arrayed longitudinally in myofibrils, being especially prominent during the formation of intercellular contacts between myocytes. Histochemically identified adult peripheral autonomic neurons cultured alone or with myocytes displayed a variety of shapes. 0~-Tubulin staining was associated with the somata and neurites of various-shaped neurons whether cultured alone or with myocytes. Cultured neurons derived from stellate and intrinsic cardiac ganglia also exhibited staining for the general neuronal marker PGP 9.5 (protein gene product 9.5), and for specific markers of the following neurochemicals: tyrosine hydroxylase, acetylcholinesterase, choline acetyltransferase, neuropeptide Y, vasoactive intestinal peptide, calcitonin gene-related peptide, bradykinin, oxytocin, and NADPH-diaphorase. These data indicate that: (a) adult ventricular myocytes cocultured with intrathoracic neurons retain the structural properties of adult myocytes found in vivo; (b) intrinsic cardiac and extrinsic intrathoracic neurons cultured alone or with cardiomyocytes display morphological characteristics similar to those of neurons studied in situ; (c) intrinsic cardiac and intrathoracic extracardiac neurons cultured alone or with cardiomy- ocytes display a variety of morphologies (unipolar, bipolar, and multipolar), larger and more multipolar neurons being present in cultures derived from stellate versus intrinsic cardiac ganglia; (d) such cultured neurons are associated with a number of neurochemicals, more than one chemical being associated with each neuron. This model presents an excellent opportunity to study the morphology of individual peripheral Departments of 1Physiology and Biophysics and of 2Anatomy and Neurobiology, Faculty of Medicine, Darhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Received 10 October 1995 Accepted 31 January 1996 Correspondence to: Dr M. Horackova. Tel: (902) 494-2268; Fax: (902) 494-1685.