Sarah A. Locknar

Alpha smooth muscle actin distribution in cytoplasm and nuclear invaginations of connective tissue fibroblasts

Alpha smooth muscle actin (α-SMA) was recently shown to be present in mouse subcutaneous tissue fibroblasts in the absence of tissue injury. In this study, we used a combination of immunohistochemistry and correlative confocal scanning laser and electron microscopy to investigate the structural organization of α-SMA in relation to the nucleus. Furthermore, we explored colocalization analysis as a method for quantifying the amount of α-SMA in close approximation to the nucleic acid marker, 4′,6-diamidino-2-phenyl-indole, dihydrochloride. Our findings indicate the presence of α-SMA within nuclear invaginations in close proximity to the nuclear membrane, but not in the nucleoplasm. Although the function of these α-SMA-rich nuclear invaginations is at present unknown, the morphology of these structures suggests their possible involvement in cellular and nuclear mechanotransduction as well as nuclear transport.

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones.

Experiments were done using guinea‐pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium‐induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca2+]i) with the Ca2+‐sensitive dye fluo‐3, we demonstrated that 10 mm caffeine activated ryanodine receptors and caused a rise in [Ca2+]i in both Ca2+‐containing and Ca2+‐deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μm thapsigargin or caffeine and 20 μm ryanodine blocked subsequent caffeine‐induced elevations of [Ca2+]i. Treatment with thapsigargin, ryanodine or 200 μm Cd2+ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μm tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca2+ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea‐pig sympathetic stellate neurones.

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. J. Comp. Neurol. 439:73–86, 2001.

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.

TRPC6 immunoreactivity is colocalized with neuronal nitric oxide synthase in extrinsic fibers innervating guinea pig intrinsic cardiac ganglia

Tachykinins depolarize guinea pig intracardiac neurons by activating nonselective cationic channels. Recently, members of the transient receptor potential family of membrane channels (TRPC) have been implicated in the generation of G protein‐coupled receptor‐activated nonselective cationic currents. We have investigated whether guinea pig cardiac neurons exhibit immunoreactivity to TRPC. Our results showed that nerve fibers within guinea pig intrinsic cardiac ganglia exhibited immunoreactivity to TRPC6. After culture of cardiac ganglia whole‐mount explants for 72 hours, the TRPC6‐IR fiber networks were absent. Therefore, the TRPC6‐IR fibers were derived from sources extrinsic to the heart. A small percentage (∼3%) of intracardiac neurons also exhibited TRPC6 immunoreactivity in control preparations, and the percentage of cells exhibiting TRPC6 immunoreactivity was not changed following explant culture for 72 hours. The few intrinsic TRPC6‐IR neurons also exhibited nitric oxide synthase (NOS) immunoreactivity, indicating that they were nitrergic as well. We compared the immunohistochemical staining patterns of TRPC6‐IR fibers with the staining patterns of a number of other neurotransmitters or neurotransmitter synthetic enzymes that mark specific extrinsic inputs to the intrinsic cardiac ganglia. The TRPC6‐IR fibers were not immunoreactive for choline acetyltransferase, tyrosine hydroxylase, or substance P. However, the TRPC6‐IR fibers exhibited immunoreactivity to neuronal NOS. Therefore, we propose that the TRPC6‐IR fibers within the guinea pig intrinsic cardiac ganglia are vagal sensory fibers that also contain NOS. We found, in support of this conclusion, that TRPC6‐IR cells were also present in sections of nodose ganglia. J. Comp. Neurol. 450:283–291, 2002.

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.

Somatic ATP release from guinea pig sympathetic neurons does not require calcium-induced calcium release from internal stores.

Prior studies indicated that a Ca(2+)-dependent release of ATP can be initiated from the soma of sympathetic neurons dissociated from guinea pig stellate ganglia. Previous studies also indicated that Ca(2+)-induced Ca(2+) release (CICR) can modulate membrane excitability in these same neurons. As Ca(2+) release from internal stores is thought to support somatodendritic transmitter release in other neurons, the present study investigated whether CICR is essential for somatic ATP release from dissociated sympathetic neurons. Caffeine increased intracellular Ca(2+) and activated two inward currents: a slow inward current (SIC) in 85% of cells, and multiple faster inward currents [asynchronous transient inward currents (ASTICs)] in 40% of cells voltage-clamped to negative potentials. Caffeine evoked both currents when cells were bathed in a Ca(2+)-deficient solution, indicating that both were initiated by Ca(2+) release from ryanodine-sensitive stores in the endoplasmic reticulum. Sodium influx contributed to generation of both SICs and ASTICs, but only ASTICs were inhibited by the presence of the P2X receptor blocker PPADs. Thus ASTICs, but not SICs, resulted from an ATP activation of P2X receptors. Ionomycin induced ASTICs in a Ca(2+)-containing solution, but not when it was applied in a Ca(2+)-deficient solution, demonstrating the key requirement for external Ca(2+) in initiating ASTICs by ionomycin. Pretreatment with drugs to deplete the internal stores of Ca(2+) did not block the ability of ionomycin or long depolarizing voltage steps to initiate ASTICs. Although a caffeine-induced release of Ca(2+) from internal stores can elicit both SICs and ASTICs in dissociated sympathetic neurons, CICR is not required for the somatic release of ATP.

Calcium Influx through Channels Other than Voltage-Dependent Calcium Channels Is Critical to the Pituitary Adenylate Cyclase-Activating Polypeptide-Induced Increase in Excitability in Guinea Pig Cardiac Neurons

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) effects on intracellular calcium ([Ca2+]i) and excitability have been studied in adult guinea pig intracardiac neurons. PACAP increased excitability, but did not elicit Ca2+ release from intracellular stores. Exposure to a Ca2+‐deficient solution did not deplete [Ca2+]i stores but did eliminate the PACAP‐induced increase in excitability. We postulate that Ca2+ influx is required for the PACAP‐induced increase in excitability.

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.