Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin.
BACKGROUND
Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated.


RESULTS
Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X(3) and TRPV1 receptors after 1-4 days in culture (together with their more frequent co-localization), while P2X(2) ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca(2+) imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist alpha, beta-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X(3) receptors (selectively antagonized by A-317491) and heteromeric P2X(2/3) receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X(3) receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X(3) receptors.


CONCLUSIONS
Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.
Neurobiology Sector, International School for Advanced Studies (SISSA), Via Beirut 4, 34014 Trieste, Italy. simonett@sissa.it
Manuela
M
Simonetti
Alessandra
A
Fabbro
Marianna
M
D'Arco
Marina
M
Zweyer
Andrea
A
Nistri
Rashid
R
Giniatullin
Elsa
E
Fabbretti
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D005724:Ganglia / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D020932:Nerve Growth Factor / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058469:Receptors, Purinergic P2X; D018689:Sensory System Agents / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000378:metabolism*; D014276:Trigeminal Nerve / Q000378:metabolism*
D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D018689:Sensory System Agents; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; C493922:Trpv1 protein, rat; D012701:Serotonin; D020932:Nerve Growth Factor; D002211:Capsaicin; D002118:Calcium