N-Type Ca²⁺ Channels in Cultured Rat Sphenopalatine Ganglion Neurons: An Immunohistochemical and Electrophysiological Study

Abstract

Summary:Results from pharmacological studies have sug gested that presynaptic N-type Ca 2 + channels play an important role in regulating neuronal Ca 2 + influx and transmitter nitric oxide (NO) release in isolated cerebral arteries. However. the presence of N-type Ca 2 + channels in cerebral perivascular nerves has not been directly demonstrated. As a major source of cerebral perivascular NOergic innervation is the sphenopalatine ganglion (SPG), adult rat SPGs were cultured and examined by whole-cell patch-clamp technique. One week after growing in the culture medium, significant neurite outgrowth from the SPG neuronal cells was observed. Both soma and neurites of these cells were immunoreactive for N-type Ca 2 + channels, transmitter-synthesizing enzymes (choline acetyl transferase and NO synthase), and several neuropeptides (vasoactive in testinal peptide, neuropeptide Y, calcitonin gene-related pep tide, substance P, and pituitary adenylate cyclase-activating Parasympathetic innervation has been shown to play a significant role in cerebral vasodilation under physi ological and pathophysiological conditions (Seylaz et aI., 1988;Suzuki and Hardebo, 1993; Kano et aI., 1991; Morita-Tsuzuki et aI., 1993;Branston, 1995;Faraci and Heistad, 1998). Parasympathetic nerves to cerebral cir culation were classically defined as cholinergic in nature 183peptide-38) that had been found in cerebral perivascular nerves in whole-mount vascular preparations. In current-clamp record ings, injection of a small depolarizing current caused action potential firing. In voltage-clamp recordings, the fast inward currents were blocked by tetrodotoxin and outward currents by tetraethylammonium, which is typical for neurons. Most Ca 2 + currents isolated by blockade of sodium and potassium currents were blocked by w-conotoxin, indicating that N-type Ca 2 + channels are the dominant voltage-dependent Ca 2 + channels regulating Ca 2 + influx during membrane depolarization of SPG neurons. The ability to culture postganglionic SPG neurons provides an opportunity to directly study the electrophysiologi cal and pharmacological properties of these neurons.

Electrophysiological and immunofluorescence characterization of Ca2+ channels of acutely isolated rat sphenopalatine ganglion neurons

“…Under our experimental conditions, the total number of isolated neurons per animal was 1213 ± 134 (SEM; n=3). This yield is greater than that reported in a previous study (< 100 neurons/rat) employing SPG [15]. In the first set of experiments, immunofluorescence assays were employed to determine the neurochemical phenotype of the acutely dissociated SPG neurons.…”

“…The yield of neurons obtained was approximately 10-fold higher than previously reported [15]. Also, the neurons were not kept in culture for long periods (< 24 hr) in order to avoid growth of processes that would affect clamp control in our experiments.…”

Cholinesterase Inhibitor Blockade and its Prevention by Statins of Sympathetic α7-nAChR-Mediated Cerebral Nitrergic Neurogenic Vasodilation

“…Freshly dissected SCGs from animals were placed in cold Hibernate A (Invitrogen, Carlsbad, CA, USA) solution (Liu et al, 2000). After being cut into smaller pieces, the ganglia were transferred to Mg 2 + /Ca 2 + -free Hanks'-balanced salt solution containing papain (2 U/ml; Sigma-Aldrich, St Louis, MO, USA), collagenase D (1.2 mg/ml; Roche Diagnostics, Indianapolis, IN, USA), and dispase (4.8 mg/ml; Invitrogen), and were incubated for 50 mins at 371C.…”

An Immunohistochemical Study of Cocaine- and Amphetamine-Regulated Transcript (Cart) Expression in the Pterygopalatine Ganglion of the Pig

“…Previous studies have also shown that PPG parasympathetic neurons are predominantly cholinergic by nature, however the presence of other neurotransmitter markers including tyrosine hydroxylase or nitric oxide synthase has been additionally noted [7]. Besides, major neurotransmitter PPG neurons also express various neuropeptide receptors [8] as well as a bulk of neuroactive peptides acting as neuromodulators and/or co-transmitters [9][10][11]. Recently, the presence of novel multipotent neuropeptide cocaine-and amphetamineregulated transcript (CART) has been found in a small subset of the rat's [12] and the duck's [13] PPG neurons, however the full meaning of this fi nding is still obscure.…”