Caudal fourth ventricular administration of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside regulates glucose and counterregulatory hormone profiles, dorsal vagal complex metabolosensory neuron function, and hypothalamic Fos expression.

This study investigated the hypothesis that estrogen controls hindbrain AMP-activated protein kinase (AMPK) activity and regulation of blood glucose, counterregulatory hormone secretion, and hypothalamic nerve cell transcriptional status. Dorsal vagal complex A2 noradrenergic neurons were laser microdissected from estradiol benzoate (E)- or oil (O)-implanted ovariectomized female rats after caudal fourth ventricular (CV4) delivery of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR), for Western blot analysis. E advanced AICAR-induced increases in A2 phospho-AMPK (pAMPK) expression and in blood glucose levels and was required for augmentation of Fos, estrogen receptor-α (ERα), monocarboxylate transporter-2, and glucose transporter-3 protein in A2 neurons and enhancement of corticosterone secretion by this treatment paradigm. CV4 AICAR also resulted in site-specific modifications in Fos immunolabeling of hypothalamic metabolic structures, including the paraventricular, ventromedial, and arcuate nuclei. The current studies demonstrate that estrogen regulates AMPK activation in caudal hindbrain A2 noradrenergic neurons during pharmacological replication of energy shortage in this area of the brain, and that this sensor is involved in neural regulation of glucostasis, in part, through control of corticosterone secretion. The data provide unique evidence that A2 neurons express both ERα and -ß proteins and that AMPK upregulates cellular sensitivity to ERα-mediated signaling during simulated energy insufficiency. The results also imply that estrogen promotes glucose and lactate uptake by these cells under those conditions. Evidence for correlation between hindbrain AMPK and hypothalamic nerve cell genomic activation provides novel proof for functional connectivity between this hindbrain sensor and higher order metabolic brain loci while demonstrating a modulatory role for estrogen in this interaction.

Expanding role of ATP as a versatile messenger at carotid and aortic body chemoreceptors.

In mammals, peripheral arterial chemoreceptors monitor blood chemicals (e.g. O(2), CO(2), H(+), glucose) and maintain homeostasis via initiation of respiratory and cardiovascular reflexes. Whereas chemoreceptors in the carotid bodies (CBs), located bilaterally at the carotid bifurcation, control primarily respiratory functions, those in the more diffusely distributed aortic bodies (ABs) are thought to regulate mainly cardiovascular functions. Functionally, CBs sense partial pressure of O(2) ( ), whereas ABs are considered sensors of O(2) content. How these organs, with essentially a similar complement of chemoreceptor cells, differentially process these two different types of signals remains enigmatic. Here, we review evidence that implicates ATP as a central mediator during information processing in the CB. Recent data allow an integrative view concerning its interactions at purinergic P2X and P2Y receptors within the chemosensory complex that contains elements of a 'quadripartite synapse'. We also discuss recent studies on the cellular physiology of ABs located near the aortic arch, as well as immunohistochemical evidence suggesting the presence of pathways for P2X receptor signalling. Finally, we present a hypothetical 'quadripartite model' to explain how ATP, released from red blood cells during hypoxia, could contribute to the ability of ABs to sense O(2) content.

Confocal immunofluorescence study of rat aortic body chemoreceptors and associated neurons in situ and in vitro.

Aortic bodies (ABs) are putative peripheral arterial chemoreceptors, distributed near the aortic arch. Though presumed to be analogous to the well-studied carotid bodies (CBs), their anatomical organization, innervation, and function are poorly understood. By using multilabel confocal immunofluorescence, we investigated the cellular organization, innervation, and neurochemistry of ABs in whole mounts of juvenile rat vagus and recurrent laryngeal (V-RL) nerves and in dissociated cell culture. Clusters of tyrosine hydroxylase-immunoreactive (TH-IR) glomus cells were routinely identified within these nerves. Unlike the CB, many neuronal cell bodies and processes, identified by peripherin (PR) and neurofilament/growth-associated protein (NF70/GAP-43) immunoreactivity, were closely associated with AB glomus clusters, especially near the V-RL bifurcation. Some neuronal cell bodies were immunopositive for P2X2 and P2X3 purinoceptor subunits, which were also found in nerve terminals surrounding glomus cells. Immunoreactivity against the vesicular acetylcholine transporter (VAChT) was detected in local neurons, glomus cells, and apposed nerve terminals. Few neurons were immunopositive for TH or neuronal nitric oxide synthase. A similar pattern of purinoceptor immunoreactivity was observed in tissue sections of adult rat V-RL nerves, except that glomus cells were weakly P2X3-IR. Dissociated monolayer cultures of juvenile rat V-RL nerves yielded TH-IR glomus clusters in intimate association with PR- or NF70/GAP-43-IR neurons and their processes, and glial fibrillary acidic protein-IR type II (sustentacular) cells. Cocultures survived for several days, wherein neurons expressed voltage-activated ionic currents and generated action potentials. Thus, this coculture model is attractive for investigating the role of glomus cells and local neurons in AB function.

Interactions of carotid sinus or aortic input with emetic signals from gastric afferents and vestibular system.

This study investigated how baro- and chemoreceptor afferents interact with emetic signals from gastric afferents and the vestibular system, and how these interactions modulate emetic and prodromal responses. We performed splanchnic denervation and abdominal vagotomy in anesthetized shrews (Suncus murinus), and then induced emetic responses by gastric distension. Next, we investigated the effects of these gastric afferent sections on cardiovascular and emetic responses induced by electrical stimulation of the aortic depressor nerve (ADN) and the carotid sinus nerve (CSN) with or without gastric distension. Splanchnic denervation abolished the prodromal response before retching and aortic baroreflex inhibition caused by gastric distension, but had no effects on the emetic response. In contrast, abdominal vagotomy abolished the emetic response induced by gastric distension with or without CSN stimulation, but without affecting gastric distension-induced or CSN stimulation-induced vascular and respiratory responses. In conscious animals, CSN denervation significantly suppressed veratrine- and motion-induced emetic responses, whereas ADN denervation had no significant effects. These results suggest that aortic baroreflex inhibition via the activation of splanchnic afferents contributes to the prodromal response before retching and circulatory homeostasis. In contrast, carotid sinus inputs, which are usually non-emetic signals, interact with vagal and vestibular inputs, and modulate the development of retching and vomiting.

Brain-derived neurotrophic factor-immunoreactive neurons in the rat vagal and glossopharyngeal sensory ganglia; co-expression with other neurochemical substances.

Immunohistochemistry for brain-derived neurotrophic factor (BDNF) was performed on the rat vagal and glossopharyngeal sensory ganglia. In the jugular, petrosal and nodose ganglia, 56.1+/-5.5%, 52.4+/-9.4% and 80.0+/-3.0% of sensory neurons, respectively, were immunoreactive for BDNF. These neurons were small- to medium-sized and observed throughout the ganglia. In the solitary tract nucleus, the neuropil showed BDNF immunoreactivity. A double immunofluorescence method demonstrated that BDNF-immunoreactive neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), P2X3 receptor, the capsaicin receptor (VR1) or vanilloid receptor 1-like receptor (VRL-1) in the jugular (CGRP, 43.5%; P2X3 receptor, 51.1%; VR1, 71.7%; VRL-1, 0.5%), petrosal (CGRP, 33.2%; P2X3 receptor, 58.4%; VR1, 54.2%; VRL-1, 23.3%) and nodose ganglia (CGRP, 1.8%; P2X3 receptor, 49.1%; VR1, 70.7%; VRL-1, 11.5%). The co-expression with tyrosine hydroxylase was also detected in the petrosal (2.9%) and nodose ganglia (2.2%). However, BDNF-immunoreactive neurons were devoid of parvalbumin in these ganglia. The present findings suggest that BDNF-containing vagal and glossopharyngeal sensory neurons have nociceptive and chemoreceptive functions.

Immunohistochemical evidence for species-specific coexistence of catecholamines, serotonin, acetylcholine and nitric oxide in glomus cells of rat and guinea pig aortic bodies.

The aortic bodies are small paraganglia distributed along the vagus nerve and its branches in the vicinity of the aortic arch which, like the carotid bodies, act as arterial chemoreceptors. In the rat carotid body, corelease of ATP and acetylcholine (ACh) from glomus cells is considered to be the main mechanism mediating fast hypoxic chemotransmission while dopamine, serotonin, and nitric oxide (NO) exert modulating effects. The present study was aimed at determination of the endogenous sources of serotonin, ACh and NO within rat and guinea pig aortic bodies by immunohistochemical double- and triple-labeling approaches, utilizing antibodies to serotonin, the NO and ACh synthesizing enzymes neuronal NO synthase (nNOS) and choline acetyltransferase (ChAT), respectively, as well as to the vesicular acetylcholine transporter (VAChT). Additional marker antibodies were directed against the rate-limiting enzyme of catecholamine synthesis, i.e. tyrosine hydroxylase (TH), and the vesicular protein, synaptophysin (SYN). In both species, all aortic body glomus cells were immunoreactive to serotonin and cholinergic markers. In the rat, all glomus cells were additionally catecholaminergic, as indicated by TH-immunoreactivity, whereas this applied only to a subgroup of guinea pig glomus cells. On the other hand, all guinea pig glomus cells were nNOS-immunoreactive, whereas only nerve fibers but not glomus cells exhibited nNOS-immunoreactivity in the rat. These data support the concept that the chemoexcitatory transmitters ACh and serotonin are involved in hypoxic excitation of aortic chemoreceptor terminals in both species. The production of the inhibitory modulators, dopamine and NO, however, appears to be species-specifically regulated.

Evidence to support the distal vagal ganglion as the origin of C cells of the ultimobranchial gland in the chick.

Formation and development of the ultimobranchial anlage were studied in chicken embryos by immunohistochemistry with the antibodies to class III beta-tubulin, TuJ1, human leukemic cell-line (HNK-1), and protein gene product (PGP) 9.5, all of which recognized neurons. Medial to the fourth aortic arch, the ultimobranchial anlage was formed by the extension of the ventral portion of the fourth pharyngeal pouch at 4.5 days of incubation. At 5 days of incubation, TuJ1-immunoreactive cells with long cell processes began to enter the ultimobranchial anlage, which displayed a follicle structure. At 6 days of incubation, numerous neuronal cells that were continuous with the distal vagal ganglion (nodose ganglion) and expressed immunoreactivity for TuJ1, HNK-1, and PGP 9.5 were found to be in direct contact with the peripheral portion of ultimobranchial anlage. The TuJ1 antibody reacted only with the neuronal cells, whereas the HNK-1 and PGP 9.5 antibodies reacted with both endodermal epithelial cells and the neuronal cells in the ultimobranchial anlage. Subsequently, the ultimobranchial anlage rapidly increased in size; the follicle wall was thickened and a central cavity disappeared. The TuJ1-immunoreactive cells were distributed throughout the ultimobranchial parenchyma in 7-day-old embryos. The neuronal cell streams from the distal vagal ganglion to the ultimobranchial anlage were still prominent at 8 days of incubation. Almost all parenchymal cells of the ultimobranchial glands were intensely immunoreactive for TuJ1, HNK-1, and PGP 9.5 between 10 and 16 days of incubation. These results indicate that the neuronal cells from the distal vagal ganglion enter into the ultimobranchial anlage and give rise to C cells, i.e., C cells differentiate along the neuronal lineage. During embryonic development, C cells of the chick ultimobranchial glands transiently express a number of neuronal properties.

Quantitative studies of the aortic bodies of the cat. I. Point-counting analysis of tissue components.

Electron micrographs of cat aortic bodies were submitted to point-counting analysis to determine the volume/volume densities (vv%) of general tissue components. Paraganglia, enclosed by a perineural sheath (endoneural paraganglia), exhibit a significantly higher density of specific tissue elements (type-I cells, type-II cells, nerves) than those which covered by fibroblasts (exoneural paraganglia). Additional criteria which allowed distinction of these types of aortic bodies were not evident in this study. On average, type-I cells occupied 27.8 vv% if related to the entire organ, or 35.6 vv% if related to the blood vessel-free space. Comparing our data with those reported for the carotid body, we found an about twofold amount of type-I cells within aortic bodies. The relevance of this finding is discussed with respect to the known different electrophysiological properties of carotid and aortic body arterial chemoreceptors.

Efferent fibres to the aortic bodies: a search for their cell bodies in the brainstem of the cat and rabbit.

An attempt has been made to determine where in the lower brainstem the cell bodies of nonsympathetic efferent fibres in the aortic nerve of the cat and rabbit are located. Horseradish peroxidase (HRP) was placed on the central end of the right cut aortic nerve of anaesthetized animals and, after an appropriate time, sections of the brainstem encompassing the rostral and caudal limits of the dorsal vagal motor nucleus and nucleus ambiguus were examined microscopically for retrogradely transported HRP. Cell bodies labelled by exogenous HRP were not found in any of the cats or rabbits exposed to HRP although reaction product, due to an endogenous response, was observed. Appropriate control experiments were performed to show that the sensitivity of the technique for demonstrating HRP in our hand was adequate. We conclude that the cell bodies of efferent fibres, of non sympathetic origin, in the aortic nerve are likely to be located outside the central nervous system.

Development of type I cells of the rabbit subclavian glomera (aortic bodies): a light, fluorescence and electron microscopic study.

The embryogenesis of the subclavian glomera (aortic bodies) is controversial. Past investigators have attributed the development of the Type I cells to mesodermal and/or neural elements. Based on the results of the present light microscopic, fluorescence histochemical and electron microscopic study of rabbit aortic bodies from 16 days of gestation (term:31 days) to four days postpartum, it appears that the Type I glomus cell are derived from cells of neural crest origin. The subclavian anlage is associated with cells of neural crest origin. The subclavian glomus anlage is associated with cells of vagal origin throughout its development. Evidence of Type I cell development from pre-existing mesodermal condensations is not observed. Type I cells exhibit formaldehyde-induced-fluorescence by the twentieth day of gestation. Dense-cored cytoplasmic vesicles are apparent by the sixteenth day of gestation. The number of cytoplasmic vesicles increases steadily, but the greatest increase of vesicles is observed between the twenty-eighth day of gestation and birth. Primitive Type I glomus cells exhibit abundant polysomes and rough endoplasmic reticulum indicative of synthetic activity. Nerve terminals are apparent adjacent to Type I cells by the twentieth day of gestation, but synaptogenesis does not occur until sometime between the twenty-fourth and twenty-eighth days of gestation. Abundant vascularity, characteristic of chemosensory glomera, is not achieved until the twenty-eighth day of gestation.

A quantitative histological study of the aortic nerve and the aortic body in the buffalo (Bubalus bubalis).

In the buffalo, the left aortic nerve ramifies in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis. The right aortic nerve ramifies over the dorsal and right aspects of the aorta ascendens near its origin. The histograms of myelinated fibres of both left and right aortic nerve are distinctly unimodal with peak around 4-6 micron (64.2-67.8%). The left aortic body is situated in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis, while the right aortic body is located in the tunica adventitia of the dorsal and right aspects of the aorta ascendens near its origin. The greatest sagittal section area of the left aortic body is 0.102 +/- 0.009 mm2 and that of the right aortic body is 0.041 +/- 0.002 mm2. The organ is highly vascular. The mean size of the glomus cells from the left aortic body is 7.68 +/- 0.9 micron x 9.37 +/- 0.13 micron (short diameter x long diameter), whereas the corresponding value for the right aortic body is 7.84 +/- 0.14 micron x 9.86 +/- 0.21 micron; and their density values are (11,417 +/- 301.7)/mm2 and (9,839 +/- 213.3)/mm2 respectively.