The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

The heart is lost without the hypothalamus.

Neuroanatomic and functional studies show the paraventricular (PVN) of the hypothalamus to have a central role in the autonomic control that supports cardiovascular regulation. Direct and indirect projections from the PVN preautonomic neurons to the sympathetic preganglionic neurons in the spinal cord modulate sympathetic activity. The preautonomic neurons of the PVN adjust their level of activation in response to afferent signals arising from peripheral viscerosensory receptors relayed through the nucleus tractus solitarius. The prevailing sympathetic tone is a balance between excitatory and inhibitory influences that arises from the preautonomic PVN neurons. Under physiologic conditions, tonic sympathetic inhibition driven by a nitric oxide-γ-aminobutyric acid-mediated mechanism is dominant, but in pathologic situation such as heart failure there is a switch from inhibition to sympathoexcitation driven by glutamate and angiotensin II. Angiotensin II, reactive oxygen species, and hypoxia as a result of myocardial infarction/ischemia alter the tightly regulated posttranslational protein-protein interaction of CAPON (carboxy-terminal postsynaptic density protein ligand of neuronal nitric oxide synthase (NOS1)) and PIN (protein inhibitor of NOS1) signaling mechanism. Within the preautonomic neurons of the PVN, the disruption of CAPON and PIN signaling leads to a downregulation of NOS1 expression and reduced NO bioavailability. These data support the notion that CAPON-PIN dysregulation of NO bioavailability is a major contributor to the pathogenesis of sympathoexcitation in heart failure.

Distribution and morphology of sensory and autonomic fibres in the subendocardial plexus of the rat heart.

Cardiac reflexes originating from sensory receptors in the heart ensure blood supply to vital tissues and organs in the face of constantly changing demands. Atrial volume receptors are mechanically sensitive vagal afferents which relay to the medulla and hypothalamus, affecting vasopressin release and renal sympathetic activity. To date, two anatomically distinct sensory endings have been identified which may subserve cardiac mechanosensation: end-nets and flower-spray endings. To map the distribution of atrial receptors in the subendocardial space, we have double-labelled rat right atrial whole mounts for neurofilament heavy chain (NFH) and synaptic vesicle protein 2 (SV2) and generated high-resolution maps of the rat subendocardial neural plexus at the cavo-atrial region. In order to elucidate the nature of these fibres, double labelling with synaptophysin (SYN) and either NFH, calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) was performed. The findings show that subendocardial nerve nets are denser at the superior cavo-atrial junction than the mid-atrial region. Adluminal plexuses had the finest diameters and stained positively for synaptic vesicles (SV2 and SYN), CGRP and TH. These plexuses may represent sympathetic post-ganglionic fibres and/or sensory afferents. The latter are candidate substrates for type B volume receptors which are excited by stretch during atrial filling. Deeper nerve fibres appeared coarser and may be cholinergic (positive staining for ChAT). Flower-spray endings were never observed using immunohistochemistry but were delineated clearly with the intravital stain methylene blue. We suggest that differing nerve fibre structures form the basis by which atrial deformation and hence atrial filling is reflected to the brain.

Electrophysiological characterisation of atrial volume receptors using ex vivo models of isolated rat cardiac atria.

NEW FINDINGS: What is the central question of this study? What ex vivo preparation of the rat's cavoatrial junction is efficient for characterising atrial mechanoreceptors? What is the main finding and its importance? Of four different ex vivo preparations, static pressure, flow, open and euthermic, the optimal preparation was the euthermic one and involved direct recording from the right cardiac vagal branch with a Langendorff style perfusion at 37°C. Type A receptors were most common, and appeared insensitive to stretch and sensitive to atrial contraction. Type B and intermediate receptors were not isolated at 20°C but were observed closer to 37°C. The findings may suggest that type A and B receptors utilise different molecular transduction mechanisms. ABSTRACT: Atrial volume receptors are a family of afferent neurons whose mechanically sensitive endings terminate in the atria, particularly at the cavoatrial junctions. These mechanosensors form the afferent limb of an atrial volume receptor reflex that regulates plasma volume. The prevailing functional classification of atrial receptors arose as a result of in vivo recordings in the cat and dog and were classified as type A, B or intermediate according to the timing of peak discharge during the cardiac cycle. In contrast, there have been far fewer studies of the common small laboratory mammals such as the rat. Using several ex vivo rat cavoatrial preparations, a total of 30 successful single cavoatrial mechanosensory recordings were obtained. These experiments show that the rat possesses type A, B and intermediate atrial mechanoreceptors as described for larger mammals. Recording these cavoatrial receptors proved challenging from the main vagus, but direct recording from the cardiac vagal branch greatly increased the yield of mechanically sensitive single units. In contrast to type A units, type B atrial mechanoreceptor activity was never observed at room temperature but required elevation of temperature to a more physiological range in order to be detected. The adequate stimulus for these receptors remains unclear; however, type A atrial receptors appear insensitive to direct atrial stretch when applied using a programmable positioner. The findings may suggest that type A and type B atrial receptors utilise different molecular transduction mechanisms.

The paraventricular nucleus and heart failure.

NEW FINDINGS: What is the topic of this review? This review gives an update on the cellular and molecular mechanisms within the autonomic nervous system involved in non-pathological and pathological cardiovascular regulation. What advances does it highlight? For cardiovascular homeostasis in non-pathological conditions to be maintained, discrete neural networks using specified signalling mechanisms at both cellular and molecular levels are required. In heart failure, the cell signalling protein partners CAPON and PIN decrease the bioavailability of nitric oxide by inhibiting neuronal nitric oxide synthase activity, leading to the removal of tonic neuronal inhibition. Following a myocardial infarction, pro-inflammatory cytokines in the paraventricular nucleus and the subsequent generation of reactive oxygen species, via angiotensin II activation of the angiotensin II type 1 receptor, increase neuronal excitability further, leading to sympathetic excitation. A pathological feature of heart failure is abnormal control of the sympathetic nervous system. The paraventricular nucleus of the hypothalamus (PVN) is one of the most important central sites involved in regulating sympathetic tone and is, in part, responsible for the dysregulation of the sympathetic nervous system evident in heart failure. Generation of sympathetic tone in response to fluctuations in cardiovascular regulation uses discrete anatomical pathways and neurochemical modulators. Direct and indirect projections from the PVN pre-autonomic neurons innervate the sympathetic preganglionic neurons in the spinal cord, which in turn innervate sympathetic ganglia that give rise to the sympathetic nerves. Pre-autonomic neurons of the PVN themselves receive an afferent input arising from the nucleus tractus solitarii, and viscerosensory receptors convey cardiovascular fluctuations to the nucleus tractus solitarii. The PVN contains excitatory and inhibitory neurons, whose balance determines the sympathetic tone. In non-pathological conditions, the tonic inhibition of the PVN pre-autonomic neurons is mediated by GABA- and NO-releasing neurons. In heart failure, the pre-autonomic neurons are disinhibited by the actions of the excitatory neurotransmitters glutamate and angiotensin II, leading to increased sympathetic activity. A key feature of the disinhibition is a reduction in the bioavailability of NO as a consequence of disrupted CAPON and PIN signalling mechanisms within the neuron. Another critical feature that contributes to increased neuronal excitation within the PVN is the production of pro-inflammatory cytokines immediately following a myocardial infarction, the activation of the angiotensin II type 1 receptor and the production of reactive oxygen species. By examining the changes associated with the sympathetic nervous system pathway, we will progress our understanding of sympathetic regulation in heart failure, identify gaps in our knowledge and suggest new therapeutic strategies.

A novel low temperature transcutaneous energy transfer system suitable for high power implantable medical devices: performance and validation in sheep.

Transcutaneous energy transfer (TET) systems use magnetic fields to transfer power across the skin without direct electrical connectivity. This offers the prospect of lifetime operation and overcomes risk of infection associated with wires passing through the skin. Previous attempts at this technology have not proved suitable due to poor efficiency, large size, or tissue damage. We have developed a novel approach utilizing frequency control that allows for wide tolerance in the alignment between internal and external coils for coupling variations of 10 to 20 mm, and relatively small size (50 mm diameter, 5 mm thickness). Using a sheep experimental model, the secondary coil was implanted under the skin in six sheep, and the system was operated to deliver a stable power output to a 15 W load continuously over 4 weeks. The maximum surface temperature of the secondary coil increased by a mean value of 3.4 +/- 0.4 degrees C (+/-SEM). The highest absolute mean temperature was 38.3 degrees C. The mean temperature rise 20 mm from the secondary coil was 0.8 +/- 0.1 degrees C. The efficiency of the system exceeded 80% across a wide range of coil orientations. Histological analysis revealed no evidence of tissue necrosis or damage after four weeks of operation. We conclude that this technology is able to offer robust transfer of power to implantable devices without excess heating causing tissue damage.

The role of free radicals in the muscle vasodilatation of systemic hypoxia in the rat.

Muscle vasodilatation evoked by systemic hypoxia is adenosine mediated and nitric oxide (NO) dependent: recent evidence suggests the increased binding of NO at complex IV of endothelial mitochondria when O(2) level falls leads to adenosine release. In this study on anaesthetised rats, the increase in femoral vascular conductance (FVC) evoked by systemic hypoxia (breathing 8 % O(2) for 5 min) was reduced by oxypurinol which inhibits xanthine oxidase (XO): XO generates O(2)(-) from hypoxanthine, a metabolite of adenosine. By contrast, infusion of superoxide dismutase (SOD), which dismutes O(2)(-) to hydrogen peroxide (H(2)O(2)), potentiated the hypoxia-evoked increase in FVC. However, NO synthesis inhibition reduced the hypoxia-evoked increase in FVC and it was not further altered by SOD. In other studies, the spinotrapezius muscle was pre-loaded with hydroethidine (HE), or dihydrorhodamine (DHR) which fluoresce in the presence of O(2)(-) and H(2)O(2), respectively. In muscle loaded with HE, systemic hypoxia increased fluorescence in endothelial cells of arterioles, whereas in muscle loaded with DHR, fluorescence was diffusely located in and around arteriolar endothelium. We propose that in systemic hypoxia, O(2)(-) generated by the XO degradation pathway from adenosine released by endothelial cells, and released by endothelial mitochondria by increased binding of NO to complex IV, is dismuted to H(2)O(2), which facilitates hypoxia-induced dilatation.

Spinal cord interneurones labelled transneuronally from the adrenal gland by a GFP-herpes virus construct contain the potassium channel subunit Kv3.1b.

Interneurones in the spinal cord are likely to play an important role in the generation of activity in sympathetic preganglionic neurones (SPNs) and, therefore, sympathetic outflow. Although the properties of these interneurones have rarely been studied directly, here we show that neurones antecedent to SPNs contain the voltage-gated potassium channel subunit Kv3.1b, while SPNs do not. SPNs and interneurones were labelled by injection of a green fluorescent protein expressing herpes simplex virus (HSV-GFP) into the adrenal gland. SPNs identified by concomitant tracing with Fluorogold did not contain Kv3.1b immunoreactivity. Significantly, neurones that did not contain Fluorogold and which were unlikely to be SPNs were double labelled for Kv3.1b and GFP. This indicates that spinal cord intemeurones antecedent to SPNs contain Kv3.1b. To test the role of Kv3.1b whole cell patch clamp recordings were made from SPNs and interneurones in spinal cord slices. Selective blockade of Kv3.1b containing channels with 30 microM 4-amino-pyridine (4-AP) or 500 microM tetraethylammonium chloride (TEA) revealed that this Kv subunit contributes to fast repolarisation and fast firing frequencies of interneurones in the vicinity of the IML, allowing them to fire action potentials at much higher frequencies than SPNs. This is the first time that transneuronal labelling with this viral construct has been combined with immunohistochemical detection of ion channels. In conjunction with our electrophysiological data, this highlights a role for the Kv3.1b subunit in shaping the activity of intemeurones involved in sympathetic control.