Neuropeptide content and physiological properties of rat cartilage- projecting sensory neurones co-cultured with perichondrial cells Karin Edoff * , Bjo ¨ rn Granseth Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linko ¨ping University, 581 58 Linko ¨ping, Sweden Received 4 September 2001; received in revised form 2 October 2001; accepted 2 October 2001 Abstract In young rats the cartilaginous epiphyses forming the knee joint are supplied with blood vessels and peptidergic sensory nerve fibres through the perichondrium and cartilage canals. In the present study we show that cartilage-related dorsal root ganglion neurones co-cultured with perichondrial cells develop extensive neurite trees and express calcito- nin gene-related peptide (CGRP) and substance P (SP) in in vivo-like proportions using retrograde tracing and immu- nohistochemistry. Moreover, whole cell patch clamp recordings from these cells showed that the majority is depolarised by application of H 1 -ions. These results are compatible with the hypothesis that a local imbalance of blood flow and metabolism during normal skeletal maturation may cause tissue acidosis eliciting release of CGRP/SP from sensory nerve endings. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Cartilage; Development; Dorsal root ganglion; Cell culture; Neuropeptides; Immunohistochemstry; Whole cell patch clamp; Protons In the rat, the cartilaginous epiphyses of the long limb bones grow rapidly before onset of secondary ossification. Growing cartilage is supplied by perichondrial blood vessels and vascularised cartilage canals occur in larger epiphyses. Moreover, peptidergic sensory nerve fibres are present in the perichondrium and in cartilage canals [7,19]. A large proportion of the dorsal root ganglion (DRG) neurones projecting to the perichondrium and cartilage canals of rat distal femoral epiphyses contain calcitonin gene-related peptide (CGRP) and/or substance P (SP) [6]. Skeletal growth in young rats is reduced by denervation [7] and theoretically, DRG neurones projecting to cartilage might promote chondrocyte proliferation and/or metabolism directly via neuropeptide release. It is also possible that CGRP and SP, which have strong effects on blood vessels [10] influence chondrocytes indirectly through vascular actions. Interestingly, many DRG neurones possess proton-gated Na 1 -channels [20] and can be excited by H 1 -ions [4]. Accordingly low pH causes release of CGRP from nerve endings in situ [4] and from DRG neurones in vitro [2]. Thus, peptidergic sensory nerve terminals release CGRP and SP in response to local tissue acidosis [4] and the resultant vasodilatation [10] and angiogenesis [9] may, conceivably, restore homeostasis. In the present study we examine the CGRP- and SP-content and some electrophy- siological properties, including proton sensitivity, of identi- fied cartilage-projecting rat DRG neurones co-cultured with perichondrial cells. The local ethics committee approved the experiments. Chemicals were purchased from Sigma (St Louis, USA) unless otherwise stated. Sprague–Dawley rat pups of both sexes (6–9 days, n ¼ 42) were used. For tracer injection, 23 animals were anaesthetised with Ketalar w (intraperitoneally (i.p.), 60 mg/kg, ketamin, Parke Davies, UK) and Domitor w (i.p., 0.5 mg/kg, medetomidin, Orion Corporation, USA). Bilateral injections of the retrograde tracer fast blue (FB, 0.2 ml) into the distal femoral epiphysis were performed as described [6]. For perichondrial cell culture, femoral and tibial epiphyses forming the knee were dissected from decapitated rat pups (n ¼ 20) and digested in 0.12% collagenase type II (1 h, 378C). After two centrifugations (1600 £ g) and washes in Dulbecco’s modified Eagles medium (DMEM) the cells were plated in plastic dishes (35 mm, 100 000 cells/dish, Nunc, Sweden) and subcultured up to five times. All cells were maintained in DMEM (378C) with 10% fetal calf serum and 1% kanamycin in 5% CO 2 Neuroscience Letters 315 (2001) 141–144 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S0304-3940(01)02353-9 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 146-13-222796; fax: 146-13- 223192. E-mail address: kared@mcb.liu.se (K. Edoff).