4. At later times (stages 25-40), the inward current of the action potential is carried by both Na+ and Ca2+: the action potential has two components, an initial spike which is blocked by removal of Na+ or addition of TTX, followed by a plateau which is blocked by La3+, Co2+ or Mn2+.5. Finally (stages 40-51), the inward current is primarily carried by Na+, since the action potential is blocked only by removal of Na+ or addition of TTX, and the overshoot agrees with the prediction of the Nernst equation for a Na-selective membrane. When the outward current channel is blocked and cells exposed to Na-free solutions, 67 % of cells at
Sensory neurons were dissociated from trigeminal ganglia or from dorsal root ganglia of rats, grown in culture, and examined for expression of properties of pain sensory cells. Many sensory neurons in culture are excited by low concentrations of capsaicin, reportedly a selective stimulus for pain sensory neurons. Many are excited by bradykinin, sensitized by prostaglandin E2, or specifically stained by an antiserum against substance P.These experiments provide a basis for the study of pain mechanisms in cell culture.Pain sensory neurons have been identified as a distinct class of sensory neurons in mammals (1-4). Pain sensory endings are activated or sensitized by painful mechanical stimulation, by painful heat, and by compounds that are released locally in damaged tissue (1,5,6). Bradykinin, prostaglandins, and amines are among the compounds that have been shown to activate or sensitize pain endings and are thought to have a role in the pain associated with injury and inflammation (5, 6).The action of these compounds on sensory endings has been studied in experimental animals (7-14), but the studies have encountered technical limitations. A major limitation is that the mechanisms that underlie excitation or sensitization ofpain sensory endings are not accessible to biophysical measurements. Other limitations are that the concentration of bradykinin, prostaglandins, or amines at the sensory endings is not accurately known; and that each of these compounds produces inflammatory changes in the tissue as well as release of other mediators, so that its actions are not evaluated in isolation. These difficulties would be alleviated if differentiated pain sensory neurons could be studied in cell culture. This alternative approach would allow more detailed pharmacological, biophysical, and biochemical studies of pain sensory neurons.As a first step toward the study ofpain mechanisms in culture, we have tested whether some characteristics of pain sensory neurons are expressed by sensory neurons in culture. We find that sensory cells grown in the absence of other cells express sensitivity to capsaicin (8-methyl-N-vanillyl-6-nonenamide), a property restricted to unmyelinated pain sensory fibers in adult animals (15-18), and in addition express other properties (7,8,10,11,19) of differentiated pain sensory neurons. METHODS Cell Culture. The portion of the trigeminal ganglion associated with the mandibular nerve was dissected from newborn rats (CD strain; Charles River Breeding Laboratories) and the cells were dissociated by treatment with dispase (grade 2; Boehringer Mannheim) and collagenase (type I; Worthington). Cells were plated on islands of collagen less than 1 mm in diameter (20) and grown in a modified L-15-CO2 growth medium (21) from which methocel and bovine serum albumin were omitted and in which glucose, penicillin, and streptomycin concentrations were reduced by half. Cultures were treated with 10 tkM 1-f3-D-arabinofuranosylcytosine (cytosine arabinoside) during the 4 days after plating to minimize gro...
SUMMARY1. Neurones of the nodose ganglion of the vagus nerve were dissociated from new-born rats and grown in the virtual absence of non-neuronal cells and in the presence of nerve growth factor.2. The resting potentials of the neurones ranged from -40 to -80 mV. Action potentials were of short duration, with no inflexion on the falling phase; others were of longer duration with a hump on the falling phase.3. The inward current of the action potential was carried either predominantly by Na+ or by Na+ and Ca2+.4. Tetrodotoxin (1 PM) blocked the Na+ channels of some neurones but in other neurones the Na+ channels were partially or completely resistant to tetrodotoxin (1-10 /M).5. Many neurones formed excitatory synapses on neighbouring neurones which were blocked or greatly reduced by conventional ganglionic nicotinic antagonists. This indicates that these neurones secreted ACh and expressed ACh receptors at these synapses.6. The accompanying paper (Baccaglini & Cooper, 1981) reports the effect of co-culturing nodose neurones with non-neuronal cells on the expression of functional nicotinic receptors.
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