Electrophysiological effects of nicotinic and electrical stimulation of intrinsic cardiac ganglia in the absence of extrinsic autonomic nerves in the rabbit heart.

BACKGROUND: The intrinsic cardiac nervous system is a rich network of cardiac nerves that converge to form distinct ganglia and extend across the heart and is capable of influencing cardiac function. OBJECTIVE: The goals of this study were to provide a complete picture of the neurotransmitter/neuromodulator profile of the rabbit intrinsic cardiac nervous system and to determine the influence of spatially divergent ganglia on cardiac electrophysiology. METHODS: Nicotinic or electrical stimulation was applied at discrete sites of the intrinsic cardiac nerve plexus in the Langendorff-perfused rabbit heart. Functional effects on sinus rate and atrioventricular conduction were measured. Immunohistochemistry for choline acetyltransferase (ChAT), tyrosine hydroxylase, and/or neuronal nitric oxide synthase (nNOS) was performed using whole mount preparations. RESULTS: Stimulation within all ganglia produced either bradycardia, tachycardia, or a biphasic brady-tachycardia. Electrical stimulation of the right atrial and right neuronal cluster regions produced the largest chronotropic responses. Significant prolongation of atrioventricular conduction was predominant at the pulmonary vein-caudal vein region. Neurons immunoreactive (IR) only for ChAT, tyrosine hydroxylase, or nNOS were consistently located within the limits of the hilum and at the roots of the right cranial and right pulmonary veins. ChAT-IR neurons were most abundant (1946 ± 668 neurons). Neurons IR only for nNOS were distributed within ganglia. CONCLUSION: Stimulation of intrinsic ganglia, shown to be of phenotypic complexity but predominantly of cholinergic nature, indicates that clusters of neurons are capable of independent selective effects on cardiac electrophysiology, therefore providing a potential therapeutic target for the prevention and treatment of cardiac disease.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Novel G protein-coupled oestrogen receptor GPR30 shows changes in mRNA expression in the rat brain over the oestrous cycle.

Oestrogen influences autonomic function via actions at classical nuclear oestrogen receptors α and ß in the brain, and recent evidence suggests the orphan G protein-coupled receptor GPR30 may also function as a cytoplasmic oestrogen receptor. We investigated the expression of GPR30 in female rat brains throughout the oestrous cycle and after ovariectomy to determine whether GPR30 expression in central autonomic nuclei is correlated with circulating oestrogen levels. In the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM) and periaqueductal gray (PAG) GPR30 mRNA, quantified by real-time PCR, was increased in proestrus and oestrus. In ovariectomised (OVX) rats, expression in NTS and VLM appeared increased compared to metoestrus, but in the hypothalamic paraventricular nucleus and PAG lower mRNA levels were seen in OVX. GPR30-like immunoreactivity (GPR30-LI) colocalised with Golgi in neurones in many brain areas associated with autonomic pathways, and analysis of numbers of immunoreactive neurones showed differences consistent with the PCR data. GPR30-LI was found in a variety of transmitter phenotypes, including cholinergic, serotonergic, catecholaminergic and nitrergic neurones in different neuronal groups. These observations support the view that GPR30 could act as a rapid transducer responding to oestrogen levels and thus modulate the activity of central autonomic pathways.

Differential distribution of 5-HT 1A and 5-HT 1B-like immunoreactivities in rat central nucleus of the amygdala neurones projecting to the caudal dorsomedial medulla oblongata.

Neurones in the central nucleus of the amygdala (CeA) projecting to the caudal dorsomedial medulla oblongata play a key role in the autonomic expression of emotional behaviour. We have earlier shown that these projections from the CeA contain gamma-aminobutyric acid. The CeA receives a dense serotonergic innervation from the raphé nuclei and expresses several serotonin (5-hydroxytryptamine, 5-HT) receptor subtypes, including the 5-HT(1A) and 5-HT(1B) subtypes. However, there have been no reports on the cellular distribution of these receptors in CeA projection neurones. This study was therefore designed to investigate the localisation of 5-HT(1A) and 5-HT(1B) receptors in CeA projection neurones identified by microinjection of a retrograde tracer, cholera toxin B-subunit (CTb) into the caudal dorsomedial medulla, targeting projections to the nucleus of the solitary tract. A large proportion (approximately 60%) of amygdala neurones retrogradely-labelled with CTb expressed 5-HT(1B) receptor-like immunoreactivity, whereas fewer (approximately 30%) expressed 5-HT(1A) receptor-like immunoreactivity. The retrogradely-labelled neurones positive for 5-HT(1B) receptor were present in both lateral and medial parts of the CeA whereas 5-HT(1A) receptor positive neurones were located mainly in the lateral part of the CeA. Since 5-HT plays an important role in controlling emotional behaviour and the 5-HT(1A) and 5-HT(1B) receptors have been shown to have distinct roles in the regulation of anxiety and depression, the differential expression of these receptors in CeA neurones projecting to the caudal dorsomedial medulla suggests that these projection neurones may act differentially in controlling autonomic expression of emotional behaviour.

Oestrogen receptors in the central nervous system and evidence for their role in the control of cardiovascular function.

Oestrogen is considered beneficial to cardiovascular health through protective effects not only on the heart and vasculature, but also on the autonomic nervous system via actions on oestrogen receptors. A plethora of evidence supports a role for the hormone within the central nervous system in modulating the pathways regulating cardiovascular function. A complex interaction of several brainstem, spinal and forebrain nuclei is required to receive, integrate and co-ordinate inputs that contribute appropriate autonomic reflex responses to changes in blood pressure and other cardiovascular parameters. Central effects of oestrogen and oestrogen receptors have already been demonstrated in many of these areas. In addition to the classical nuclear oestrogen receptors (ERalpha and ERbeta) a recently discovered G-protein coupled receptor, GPR30, has been shown to be a novel mediator of oestrogenic action. Many anatomical and molecular studies have described a considerable overlap in the regional expression of these receptors; however, the receptors do exhibit specific characteristics and subtype specific expression is found in many autonomic brain areas, for example ERbeta appears to predominate in the hypothalamic paraventricular nucleus, whilst ERalpha is important in the nucleus of the solitary tract. This review provides an overview of the available information on the localisation of oestrogen receptor subtypes and their multitude of possible modulatory actions in different groups of neurochemically and functionally defined neurones in autonomic-related areas of the brain.

Central nucleus of amygdala projections to rostral ventrolateral medulla neurones activated by decreased blood pressure.

The central nucleus of amygdala (CeA) participates in cardiovascular regulation during emotional behaviour but it has not been established whether any of these effects are mediated through its direct connections to blood pressure-regulating neurones in the rostral ventrolateral medulla (RVLM). The RVLM contains barosensitive neurones that maintain resting blood pressure via their projections to sympathetic preganglionic neurones in the thoracic spinal cord. In this study on rats, we used combined anterograde neuronal tracing of CeA projections with confocal and electron microscopic immunohistochemical detection of phenylethanolamine-N-methyltransferase, the adrenaline-synthesizing enzyme present in C1 catecholamine neurones of the RVLM, and Fos, the protein product of the c-fos proto-oncogene. Fos expression in barosensitive neurones was stimulated by an intravenous infusion of the hypotensive agent sodium nitroprusside. Injection of the tracer biotin dextran amine (10-kDa form) into the CeA resulted in anterograde labelling of axons and varicosities throughout the RVLM without retrograde labelling of somata in any brain area. With confocal microscopy, presumptive CeA terminals were found in close apposition to adrenergic (phenylethanolamine-N-methyltransferase-immunoreactive) and non-adrenergic neurones that displayed Fos-immunoreactive nuclei in response to decreased blood pressure. Electron microscopic analysis confirmed that some labelled terminals of CeA axons made synaptic contact with c-fos-activated adrenergic neurones. The results provide evidence that cardiovascular influences elicited from the CeA during stressful events may be mediated, at least in part, via monosynaptic neural projections to barosensitive sympathetic blood pressure-regulating neurones in the RVLM.

Substance P (NK1) and somatostatin (sst2A) receptor immunoreactivity in NTS-projecting rat dorsal horn neurones activated by nociceptive afferent input.

Spinal neurones that receive inputs from primary afferent fibres and have axons projecting supraspinally to the medulla oblongata may represent a pathway through which nociceptive and non-nociceptive peripheral stimuli are able to modulate cardiorespiratory reflexes. Expression of the neurokinin-1 (NK1) receptor is believed to be an indicator of lamina I cells that receive nociceptive inputs from substance P releasing afferents, and similarly, sst2A receptor expression may be a marker for neurones receiving somatostatinergic inputs. In this study, immunoreactivity for these two receptors was investigated in rat spinal neurones retrogradely labelled by injections of cholera toxin B or Fluorogold into the nucleus of the solitary tract (NTS). In addition, nociceptive activation of these labelled cells was studied by immunodetection of Fos protein in response to cutaneous and visceral noxious chemical stimuli. NK1 and sst2A receptors in lamina I were localised to mainly separate populations of retrogradely labelled cells with fusiform, flattened and pyramidal morphologies. Examples of projection neurones expressing both receptors were, however observed. With visceral stimulation, many retrogradely labelled cells expressing c-fos were immunoreactive for the NK1 receptor, and a smaller population was sst2A positive. In contrast, with cutaneous stimulation, only NK1 positive retrogradely labelled cells showed c-fos expression. These data provide evidence that lamina I neurones receiving noxious cutaneous and visceral stimuli via NK1 receptor activation project to NTS and so may be involved in coordinating nociceptive and cardiorespiratory responses. Moreover, a subpopulation of projection neurones that respond to visceral stimuli may receive somatostatinergic inputs of peripheral, local or supraspinal origins.

Evidence for peptide co-transmission in retrograde- and anterograde-labelled central nucleus of amygdala neurones projecting to NTS.

Synaptic terminals in the nucleus of the solitary tract (NTS) from axons originating in the central nucleus of the amygdala (CeA) are known to contain gamma-aminobutyric acid (GABA) immunoreactivity. Here, we have investigated whether such projections contain neuropeptides as putative co-transmitters. Somata in the medial and lateral CeA that were retrogradely labelled with cholera toxin B (CTb) injected into the commissural NTS were found to be immunoreactive for GABA, somatostatin (SOM), neurotensin (NT), vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS). Subpopulations of fibres in the NTS that were anterogradely labelled with biotin dextran amine (BDA) injected into the CeA and examined using both fluorescence and electron microscopy appeared to colocalise somatostatin, but not other neuropeptides. Their varicosities were observed in proximity to NTS neurones that were immunoreactive for the somatostatin receptor sst2A subtype, substance P (SP) NK1 receptor, and the GABAA receptor alpha3, beta1 and gamma2 subunits. This morphological evidence is consistent with the possibility of GABA-somatostatin co-transmission at synapses of some of the CeA projection neurones to NTS that might inhibit cardiovascular reflex responses in response to fear or emotion-related stimuli.