Cultured Nodose Ganglion Neurons Research Articles

Cultured nodose ganglia neurons co‐express thromboxane A2 receptor and TRPV1 mRNA

Previous studies from this laboratory have revealed that the arachidonic acid metabolite, thromboxane A2 (TxA2), stimulates vagal afferent nerves and elicits cardiovascular and pulmonary reflexes. Stimulation of sensory nerves by TxA2 could involve a neuronal TxA2 receptor (TP) since we have recently reported that TxA2 receptor mRNA is expressed in a subpopulation of cultured neuronal cells of the rabbit nodose ganglia. The present study was designed to further characterize the afferent vagal neurons that express TxA2 receptor mRNA. Since TxA2 is released during inflammation or trauma we hypothesize that TP is expressed in TxA2 nociceptive (pain-sensing) neurons. This hypothesis was tested using multiplex single-cell RT-PCR on cultured nodose ganglion neurons from rabbit. Individual cells were isolated from culture and placed in an RT-PCR reaction buffer containing primers for neurofilament medium (NFM), TP, and the capsaicin receptor (TRPV1). NFM was used as a positive neuronal marker, while the presence of TRPV1 was used to distinguish nociceptive neurons. 70% (55 of 79) of the NFM positive cells contained TRPV1 mRNA while 25% (20 of 79) of the NFM positive cells contained TP mRNA. Nearly two-thirds (13 of 20) of the TP positive cells were also positive for the TRPV1 mRNA. The presence of TP mRNA in TRPV1 positive neurons may indicate that TxA2 may activate nociceptive reflexes during events such as myocardial ischemia. Hanke was supported by an NIGMS IMSD project GM62232. Wacker was supported by IRACDA post doctoral fellowship GM63651

Leptin and CCK selectively activate vagal afferent neurons innervating the stomach and duodenum

The hormone leptin and the gut peptide CCK synergistically interact to enhance the process of satiation. Although this interaction may occur at several levels of the neuroaxis, our previous results indicate that leptin can specifically enhance the satiation effect of CCK by acting on subdiaphragmatic vagal afferent neurons. Because of this localized action, we hypothesized that a high proportion of vagal afferent neurons innervating the stomach or duodenum would be responsive to leptin and/or CCK. To test this hypothesis, we measured changes in cytosolic calcium levels induced by leptin and CCK in cultured nodose ganglion neurons labeled with a retrograde neuronal tracer injected into either the stomach or the duodenum. In the neurons labeled from the stomach, CCK activated 74% (39 of 53) compared with only 35% (34 of 97) of nonlabeled cells. Of the CCK-responsive neurons 60% (18 of 30) were capsaicin-sensitive. Leptin activated 42% (22 of 53) of the stomach innervating neurons compared with 26% of nonlabeled neurons. All of the leptin-sensitive neurons labeled from the stomach also responded to CCK. In the neurons labeled from the duodenum, CCK activated 71% (20 of 28). Of these CCK-responsive neurons 80% (12 of 15) were capsaicin sensitive. Leptin activated 46% (13 of 28) of these duodenal innervating neurons, of which 89% (8 of 9) were capsaicin-sensitive. Among neurons labeled from the duodenum 43% (12 of 28) were responsive to both leptin and CCK, compared with only 15% (15 of 97) of unlabeled neurons. Our results support the hypothesis that vagal afferent sensitivity to CCK and leptin is concentrated in neurons that innervate the stomach and duodenum. These specific visceral afferent populations are likely to comprise a substrate through which acute leptin/CCK interactions enhance satiation.

Detection of thromboxane A 2 receptor mRNA in rabbit nodose ganglion neurons

Thromboxane A 2 (TXA 2) is an arachidonic acid metabolite that is released during tissue trauma and elicits platelet aggregation and vascular smooth muscle contraction. Previous research has shown that TXA 2 stimulates pulmonary and cardiac vagal afferent neurons. Therefore, we hypothesized that the presence of the TXA 2 receptor (TP) in vagal neurons would allow for stimulation or modulation of these neurons by TXA 2. To test this hypothesis, single cell RT-PCR was employed using neurons obtained from primary cell cultures of nodose ganglia excised from adult rabbits. Since the sequence for the rabbit TP gene was unknown, a portion of the rabbit TP cDNA was first amplified, cloned, and sequenced. Primer sets for TP were then designed based on this sequence and used in conjunction with a neuronal marker, medium weight neurofilament (NFM), in multiplex RT-PCR reactions. Ninety-three cells were isolated from culture and RT-PCR was carried out on individual cells. Using an aliquot from the initial RT-PCR reaction, a second round of PCR was then employed in which the NFM and TP primer sets were split up into separate reactions. Twenty-three of the 82 cells that were positive for NFM were also positive for TP. Therefore, we conclude that the presence of TP mRNA in a subset of cultured nodose ganglion neurons allows for the possibility that TXA 2 may directly stimulate or modulate vagal afferent neurons.

Dibutyryl cGMP raises cytosolic concentrations of Ca 2+ in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca 2+ ([Ca 2+] i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca 2+] i in 42% of neurons ( n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca 2+ abolished the dbcGMP effect. Application of Ni 2+ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca 2+ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca 2+ channel antagonist, or L-type Ca 2+ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.

Localization and functional effects of adenosine A1 receptors on cardiac vagal afferents in adult rats.

There is evidence to suggest that during ischemia adenosine acts on cardiac vagal afferent neurons to activate systemic reflexes and to modulate cardiac nociception. The purpose of this study was to determine whether adenosine receptors are present and have direct cellular electrophysiological actions on cardiac vagal afferent neurons. In radioreceptor assays of nodose ganglion tissue from rats, binding was detectable for A1 (39.6 +/- 1.2 fmol/mg protein) but not A2a adenosine receptors. These findings were confirmed using the complementary approach of receptor-labeling autoradiography. Using in situ hybridization, we saw specific labeling over approximately 50% of neurons in the nodose ganglia, but not over nonneuronal cells. In colabeling studies, cardiac vagal afferent neurons were identified by retroneuronal labeling with fluororuby. Of cardiac vagal afferents approximately one-half were strongly positive for A1 adenosine receptors (immunocytochemistry). In patch-clamping experiments, adenosine inhibited peak inward calcium current in 7 of 11 cells by 48 +/- 13%. In conclusion, adenosine A1 receptors reside on a subset of vagal afferent neurons, including cardiac vagal afferents, and have electrophysiological effects that modulate neuroexcitability in cultured nodose ganglion neurons.

Nitric oxide raises cytosolic concentrations of Ca 2+ in cultured nodose ganglion neurons from rabbits

The effects of donors of nitric oxide (NO), namely, S-nitroso- N-acetylpenicillamine (SNAP) and E-methyl-2- E-hydroxyimino-5-nitro-6-methoxy-3-hexeneamide (NOR1), on cytosolic concentrations of Ca 2+ ([Ca 2+] i) were studied in cultured nodose ganglion neurons from newborn and young rabbits by loading with fura-2AM and microfluorometry. Application of SNAP (5 μM-1 mM) increased [Ca 2+] i in 46% of neurons tested. The threshold dose of the response was 10 μM and the response increased in a dose-dependent fashion. The increase in [Ca 2+] i at 50 μM averaged 74 ± 8% above the control value. [Ca 2+] i rose immediately after injection of SNAP and the plateau level was maintained in the presence of SNAP. NOR1, another donor of NO, increased [Ca 2+] i with an average increase of 82 ± 7% at 50 μM. Quantitation of NO gas in the solution of NOR1 by a redox chemiluminescence method revealed the constant release of 0.06 ppm NO from 5 ml of a 20 μM solution of NOR1. These data suggest that NO released from donors of NO induced an increase in [Ca 2+] i in nodose ganglion neurons and, therefore, that NO might play a role as a transmitter or a modulator in autonomic primary sensory systems.

Mechanisms of baroreceptor activation.

The determinants of the nerve activity generated at the baroreceptor endings have been examined. 1) In the isolated carotid sinus, the placement of activated bovine aortic endothelial cells decreased baroreceptor activity (BRA) in a reversible manner. Both endothelin and nitric oxide (NO) suppress BRA, whereas prostacyclin (PGI2) increases activity. 2) The BRA in single units declines and often ceases during non-pulsatile increases in carotid sinus pressure sustained over several minutes. This "adaptation" is attenuated by the transient potassium channel (IA) blocker 4-aminopyridine (4-AP) and not by inhibition of the Na+/K+ pump. 3) In preliminary studies, mechano-electrical transduction was examined in isolated and cultured nodose ganglion neurons. Opening of stretch-activated (SA) channels by suction on the cell-attached patch was seen infrequently; however, probing the neurons consistently increased their intracellular calcium [Ca++]i measured with fura-2. This increase in [Ca++]i is blocked by gadolinium (Gd3+), a trivalent lanthanide reported to block SA channels. Gd3+ also blocks the BRA in the carotid sinus. We conclude that paracrine factors significantly modulate BR sensitivity, that selective ionic mechanisms (the 4-AP sensitive K+ channels) determine the degree of "adaptation" of BR to elevated pressure, and that SA channels sensitive to Gd3+ may be the mechano-electrical transducers in BR neurons.

Different effects of removing extracellular Ca2+ on cytosolic Ca2+ response to anoxia of sensory neurons and carotid chemoreceptor cells from newborn rabbits.

To study the mechanism of the increases in cytosolic Ca2+ ([Ca2+]i) induced by anoxia, the author examined the effect of removing extracellular Ca2+ on the [Ca2+]i response in cultured nodose ganglion neurons (control) and carotid body glomus cells prepared from newborn rabbits using a fura-2 microfluorimetry. The sensory neurons showed a small increase in [Ca2+]i during anoxia which was not affected by the removal of extracellular Ca2+ or D600. On the other hand, in the chemoreceptor glomus cells, Ca2+ removal eliminated the anoxia-induced large [Ca2+]i increase in 85% of the tested cells (n = 67), and depressed it in the rest. Interestingly, recovery from exposure to an anoxic Ca(2+)-free solution reversibly produced a large and transient rise in [Ca2+]i. The magnitude of this post-anoxic/Ca(2+)-free [Ca2+]i transient correlated with the intensity of the suppression of the [Ca2+]i response to anoxia in Ca(2+)-free solution. Also the [Ca2+]i transient was mostly inhibited by L-type Ca2+ channel blockers (nifedipine and D600), but was not affected by tetrodotoxin. These results suggest that the anoxia-induced large increase in [Ca2+]i were, in most glomus cells, coming from extracellular sources and, in a few cells, from both extracellular sources and from intracellular pools, whereas the slight increase in [Ca2+]i in sensory neurons was probably produced by releasing Ca2+ from intracellular stores. The post-anoxic/Ca(2+)-free [Ca2+]i transient, seen in the chemoreceptor cells alone, may have resulted from modification of the anoxic [Ca2+]i response by Ca2+ removal and involves mostly L-type Ca2+ channels and a few other Ca2+ entry pathways.