Electrophysiological and immunofluorescence characterization of Ca(2+) channels of acutely isolated rat sphenopalatine ganglion neurons

Abstract

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.

Fig. 1

Immunofluorescence staining with antibodies targeted against acetylcholinesterase (AChE, A, B) and tyrosine hydroxylase (TH, C, D) in adult rat SPG (A, C) and SCG (B, D) neurons. Mouse antibodies to AChE and TH were employed and was followed by using Alexa Fluor 488-labeled goat anti-mouse secondary antibodies. E and F are images of SPG neurons incubated with the secondary antibody (without the primary antibody) and served as negative controls. Fluorescence images were acquired with a filter set containing an excitation filter at 480 nm and an emission filter at 535 nm at 20X. The images were pseudocolored; scale bar represents 100 @m. Insets show the phase contrast images for each field. E and F are images of SPG neurons immunostained with the secondary antibody in the absence of primary AChE and TH antibodies, respectively.

Fig. 2

Pharmacological identification of Ca²⁺ channel subtypes present in SPG neurons. (A) Time course of Ca²⁺ current amplitude recorded every 10 sec. Currents were evoked with the double-pulse protocol (B, top) in the absence (traces 1, 2, 5, 6, 9, 10, 13 and 14) and presence of 10 @M Nif (traces 3 and 4), 0.2 @M Aga (traces 7 and 8), 0.3 @M SNX (traces 11 and 12) and 10 @M Ctx (traces 15 and 16). Solid bars indicate the sequential exposure to the channel blockers. (B) Superimposed current traces from time course before (lower traces) and during (upper traces) exposure to Ca²⁺ channel blockers. Numbers accompanying traces correspond to recordings from time course in (A). (C) Summary plot of the Ca²⁺ current inhibition produced by the channel blockers. Numbers in parenthesis indicate the number of neurons tested.

Fig. 3

Effect of M₂ and M₄ muscarinic receptor blockers on the Oxo-M-mediated Ca²⁺ current inhibition, and of selective G protein-coupled receptor agonists on Ca²⁺ channel currents in rat SPG neurons. (A) Superimposed Ca²⁺ current traces from SPG neurons before (lower traces, Con) and during (upper traces) agonist application. Currents were evoked with the double-pulse voltage protocol (shown on top of 3A). Neurons were exposed to Oxo-M (10 @M, i), Oxo-M + Methoctramine (5 @M, ii), Oxo-M + M4 toxin (0.2 @M, iii), ATP (100 @M, iv), Ang II (10 @M, v) and NE (10 @M, vi). (B) Summary graph showing Ca²⁺ current inhibition (%) ± SEM in the presence of the agonists and muscarinic receptor blockers. Values in parentheses indicate the number of cells tested. * P < 0.01 compared to Oxo-M-exposed control neurons, Student’s t-test.