Identification of Mineralocorticoid Receptors, Aldosterone, and Its Processing Enzyme CYP11B2 on Parasympathetic and Sympathetic Neurons in Rat Intracardiac Ganglia

Recent interest has focused on the mineralocorticoid receptor (MR) and its impact on the myocardium and the performance of the heart. However, there is a lack of evidence about MR expression and its endogenous ligand aldosterone synthesis with specific regard to the intrinsic cardiac nervous system. Therefore, we looked for evidence of MR and aldosterone in sympathetic and parasympathetic neurons of intracardiac ganglia. Tissue samples from rat heart atria were subjected to conventional reverse-transcriptase polymerase chain reaction (PCR), Western blot, and double immunofluorescence confocal analysis of MR, corticosterone-inactivating enzyme 11β-hydroxysteroid-dehydrogenase-2 (11β-HSD2), aldosterone, and its processing enzyme CYP11B2 together with the neuronal markers vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH). Our results demonstrated MR, 11β-HSD2, and CYP11B2 specific mRNA and protein bands in rat heart atria. Double immunofluorescence labeling revealed coexpression of MR immunoreactivity with VAChT in large diameter parasympathetic principal neurons. In addition, MR immunoreactivity was identified in TH-immunoreactive small intensely fluorescent (SIF) cells and in nearby VAChT- and TH-immunoreactive nerve terminals. Interestingly, the aldosterone and its synthesizing enzyme CYP11B2 and 11β-HSD2 colocalized in MR– immunoreactive neurons of intracardiac ganglia. Overall, this study provides first evidence for the existence of not only local expression of MR, but also of 11β-HSD2 and aldosterone with its processing enzyme CYP11B2 in the neurons of the cardiac autonomic nervous system, suggesting a possible modulatory role of the mineralocorticoid system on the endogenous neuronal activity on heart performance.

The superior cervical ganglia modulate ventilatory responses to hypoxia independently of pre-ganglionic drive from the cervical sympathetic chain

Superior cervical ganglia (SCG) post-ganglionic neurons receive pre-ganglionic drive via the cervical sympathetic chain (CSC). The SCG projects to structures like the carotid bodies (e.g., vasculature, chemosensitive glomus cells), upper airway (e.g., tongue, nasopharynx) and to parenchyma and cerebral arteries throughout the brain. We previously reported that a hypoxic gas challenge elicited an array of ventilatory responses in sham-operated (SHAM) freely-moving adult male C57BL6 mice and that responses were altered in mice with bilateral transection of the cervical sympathetic chain (CSCX). Since the CSC provides pre-ganglionic innervation to the SCG, we presumed that mice with superior cervical ganglionectomy (SCGX) would respond similarly to hypoxic gas challenge as CSCX mice. However, while SCGX mice had altered responses during hypoxic gas challenge that occurred in CSCX mice (e.g., more rapid occurrence of changes in frequency of breathing and minute ventilation), SCGX mice displayed numerous responses to hypoxic gas challenge that CSCX mice did not, including reduced total increases in frequency of breathing, minute ventilation, inspiratory and expiratory drives, peak inspiratory and expiratory flows, and appearance of non-eupneic breaths. In conclusion, hypoxic gas challenge may directly activate sub-populations of SCG cells, including sub-populations of post-ganglionic neurons and small intensely fluorescent (SIF) cells, independently of CSC drive, and that SCG drive to these structures dampens the initial occurrence of the hypoxic ventilatory response, while promoting the overall magnitude of the response. The multiple effects of SCGX may be due to loss of innervation to peripheral and central structures with differential roles in breathing control.

Distribution and immunohistochemical characteristics of cocaine- and amphetamineregulated transcript-positive nerve elements in the pelvic ganglia of the female pig

Cocaine- and amphetamine-regulated transcript (CART) peptides are widely expressed not only in the brain but also in numerous endocrine/neuroendocrine cells as well as in neurons of the peripheral nervous system. The present study investigated the distribution patterns of CART-like immunoreactivity in the pelvic plexus (PP) of the female pig. The co-expression of CART with principal neurotransmitter markers: choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), serotonin (5-HT) or biologically active neuropeptides: pituitary adenylate cyclase-activating polypeptide (PACAP), substance P (SP), calbindin was analyzed using double immunohistochemical stainings. Amongst neurons immunopositive to Hu C/D panneuronal marker as many as 4.1 ± 1.2% in right and 4.4 ± 1.6% in left pelvic ganglia were found to express CART. The vast majority of CART-IR ganglionic neurons were predominantly small in size and were evenly scattered throughout particular ganglia. Immunoreactivity to CART was also detected in numerous nerve terminals (which frequently formed pericellular formations around CART-negative perikarya) as well as in numerous nerve fibres within nerve branches interconnecting the unilateral pelvic ganglia. Immunohistochemistry revealed that virtually all CART-IR neurons were cholinergic in nature and CART-IR basket-like formations frequently encircled TH-positive/CART-negative perikarya. None of CART-IR ganglionic neurons showed immunoreactivity to SP, PACAP, 5-HT or calbindin. CART-IR nerve fibres ran in a close vicinity to serotonin-containing cells or faintly labelled SP-expressing neurons. On the other hand, PACAP-IR, SP-IR (but not 5-HT-positive) nerve terminals were found to run in close proximity to CART-IR neurons. Our results indicate that: 1) CART present in PP may influence the activity of pelvic ganglionic neurons/SIF cells, 2) PP should be considered as a potential source of CART-like supply to pelvic viscera and 3) functional interactions between CART and SP or PACAP are possible at the periphery.

Neurochemical characteristics of paracervical ganglion in the pig

A study on the presence of the selected biologically active substances in nerve structures of the paracervical ganglion in the pig was performed with the use of immunofluorescence and RT-PCR. Immunohistochemical methods revealed that 23% of paracervical ganglion (PCG) neurons contain both tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH) and that the remaining 77% contain choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). 73% of TH/DβH neurons contained neuropeptide Y (NPY) and 8% contained somatostatin (Som). All ChAT/VAChT positive neurons contained vasoactive intestinal polypeptide (VIP), 87% of them contained Som, 76% contained NPY and 32% contained neuronal nitric oxide synthase (nNOS). Galanin (Gal) was found only in small cells, which were thought to be SIF cells. No pituitary adenylate cyclase-activating polypeptide (PACAP)- or substance P (SP)-positive neurons were found in PCG. Some areas of PCG contained dense plexuses of ChAT- and VAChT-positive nerve fibres. In the ganglion small number of TH-, nNOS-, NPY-, VIP-, Gal-, PACAP-, Som- and SP-positive nerve fibres was also visible. RT-PCR detected the presence of mRNA for TH, ChAT, nNOS, NPY, VIP, Gal and Som, which were visualised as clearly discernible bands on a gel. In cases of PACAP and SP only weak bands were observed.