Trophic factor modulation of cocaine- and amphetamine-regulated transcript peptide expression in explant cultured guinea-pig cardiac neurons.

The present study investigated the influence of trophic factors on the expression of cocaine- and amphetamine-regulated transcript peptide (CARTp) in guinea-pig cardiac ganglia maintained in explant culture. In acutely isolated cardiac ganglia preparations, <1% of the cholinergic cardiac neurons exhibited CARTp immunoreactivity. In contrast, this number increased to >25% of the cardiac neurons after 72 h in explant culture. This increase in the number of CARTp neurons in cultured cardiac ganglia explants was accompanied by an increase in CARTp transcript levels as assessed by real time polymerase chain reaction. Treatment of cardiac ganglia cultures with neurturin or glial-derived trophic factor (both at 10 ng/ml) for 72 h prevented the increase in neurons that exhibited CARTp immunoreactivity. In contrast, treatment with ciliary neurotrophic factor (50 ng/ml) for 72 h produced a small significant increase in the percentage of CARTp-immunoreactive cardiac neurons and treatment with nerve growth factor (100 ng/ml) had no effect. Neurturin treatment also decreased cardiac neuron CARTp levels after 72 h in explant culture. Cardiac neurons exhibited immunoreactivity to the neurturin receptor GFRalpha2 whereas non-neural cells preferentially exhibited immunoreactivity to the glial-derived neurotrophic factor receptor GFRalpha1 and neurturin transcripts were detected in cardiac tissue extracts. We hypothesize that a target-derived inhibitory factor, very likely neurturin, is a critical factor suppressing the expression of CARTp in guinea-pig cardiac neurons. These observations contrast with those reported in sympathetic neurons that suggest up-regulation of trophic factors after axotomy or during explant culture is a key factor contributing to the up-regulation of many neuropeptides.

The modulation of action potential generation by calcium-induced calcium release is enhanced by mitochondrial inhibitors in mudpuppy parasympathetic neurons.

Previously, we demonstrated that outward currents activated by calcium-induced calcium release (CICR) opposed depolarization-induced action potential (AP) generation in dissociated mudpuppy parasympathetic neurons [J Neurophysiol 88 (2002) 1119]. In the present study, we tested whether AP generation by depolarizing current ramps could be altered by dissipating the mitochondrial membrane potential and thus interrupting mitochondrial Ca2+ buffering. Exposure to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 2 microM) alone or in combination with the mitochondrial ATP synthase inhibitor oligomycin (8 microg/ml), increased the latency to AP generation. Exposure to the electron transport chain inhibitor rotenone (10 microM) alone or in combination with oligomycin (8 microg/ml) similarly increased the latency to AP generation. CCCP and oligomycin or rotenone and oligomycin treatment caused rhodamine 123 loss from mitochondria within a few minutes, confirming that the mitochondrial membrane potential was dissipated during drug exposure. Oligomycin alone had no effect on the latency to AP generation and did not cause loss of rhodamine 123 from mitochondria. The increase in latency induced by CCCP and oligomycin was similar when recordings were made with either the perforated patch or standard whole cell patch recording configuration. Exposure to the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (1 microM), decreased the latency to AP generation. In cells pretreated with thapsigargin to eliminate CICR, CCCP and oligomycin had no effect on AP latency. Pretreatment with iberiotoxin (IBX; 100 nM), an inhibitor of large conductance, calcium- and voltage-activated potassium channels, reduced the extent of the CCCP- and oligomycin-induced increase in latency to AP generation. These results indicate that treatment with CCCP or rotenone to dissipate the mitochondrial membrane potential, a condition which should minimize sequestration of Ca2+ by mitochondria, facilitated the Ca(2+)-induced Ca2+ release activation of IBX-sensitive and IBX-insensitive conductances that regulate AP generation.

Innervation of guinea-pig stellate ganglia by nitric oxide synthase, cocaine- and amphetamine-regulated transcript protein- and pituitary adenylate cyclase activating polypeptide-immunoreactive fibers.

The present study analyzed using immunohistochemical labeling the distribution and co-localization of nitric oxide synthase (NOS), cocaine- and amphetamine-regulated transcript peptide (CARTp) and pituitary adenylate cyclase activating polypeptide (PACAP) with choline acetyltransferase (ChAT)-immunoreactive fibers in the guinea-pig stellate ganglia. ChAT-immunoreactive fibers make pericellular baskets around virtually all stellate ganglia neurons. Pericellular baskets of NOS, CARTp and PACAP fibers were also present around numerous stellate ganglia neurons. Although all the NOS and PACAP fibers also exhibited ChAT immunoreactivity, only some of the CARTp fibers were ChAT-immunoreactive. No evidence of co-localization of NOS, PACAP and CARTp was obtained.These results indicate that NOS, PACAP and CARTp are present in distinct preganglionic axons innervating the guinea-pig stellate ganglia.

Distribution of cocaine- and amphetamine-regulated transcript peptide in the guinea pig intrinsic cardiac nervous system and colocalization with neuropeptides or transmitter synthetic enzymes.

This study was conducted to establish the presence of cocaine- and amphetamine-regulated transcript peptide (CARTp) immunoreactivity in neurons and fibers within guinea pig atrial whole-mount preparations containing the intrinsic cardiac ganglia. Many cardiac ganglia, but not all, in a given whole-mount preparation, were innervated by CARTp-immunoreactive (IR) fibers. Following explant culture of whole mounts for 72 hours, the CARTp-IR fiber networks were absent, but the number of CARTp-IR neurons was increased markedly. These observations suggested that the majority of the CARTp-IR fibers in the intracardiac ganglia were derived from sources extrinsic to the heart. In control whole-mount preparations, very few CARTp-positive neurons were present. The few intrinsic CARTp-IR neurons also exhibited choline acetyltransferase (ChAT) immunoreactivity, indicating that they make up a small subpopulation of cholinergic postganglionic neurons. Some CARTp-IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of CARTp-IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs. The CARTp-IR fibers were not immunoreactive for ChAT, tyrosine hydroxylase, calcitonin gene-related peptide, or substance P. However, virtually all CARTp-IR fibers exhibited immunoreactivity to neuronal NOS (a marker for nitric oxide-producing neurons). CARTp-IR cells and NOS-IR cells were present in the nodose ganglia. In addition, CARTp-IR neurons in the nodose also were stained positively for NADPH-diaphorase. Thus, we propose that most CARTp-IR fibers within the guinea pig intrinsic cardiac ganglia are vagal afferent fibers that also contain NOS.