Oxytocin and corticotropin-releasing hormone exaggerate nucleus tractus solitarii neuronal and synaptic activity following chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) in rodents mimics the hypoxia-induced elevation of blood pressure seen in individuals experiencing episodic breathing. The brainstem nucleus tractus solitarii (nTS) is the first site of visceral sensory afferent integration, and thus is critical for cardiorespiratory homeostasis and its adaptation during a variety of stressors. In addition, the paraventricular nucleus of the hypothalamus (PVN), in part through its nTS projections that contain oxytocin (OT) and/or corticotropin-releasing hormone (CRH), contributes to cardiorespiratory regulation. Within the nTS, these PVN-derived neuropeptides alter nTS activity and the cardiorespiratory response to hypoxia. Nevertheless, their contribution to nTS activity after CIH is not fully understood. We hypothesized that OT and CRH would increase nTS activity to a greater extent following CIH, and co-activation of OT+CRH receptors would further magnify nTS activity. Our data show that compared to their normoxic controls, 10 days' CIH exaggerated nTS discharge, excitatory synaptic currents and Ca2+ influx in response to CRH, which were further enhanced by the addition of OT. CIH increased the tonic functional contribution of CRH receptors, which occurred with elevation of mRNA and protein. Together, our data demonstrate that intermittent hypoxia exaggerates the expression and function of neuropeptides on nTS activity. KEY POINTS: Episodic breathing and chronic intermittent hypoxia (CIH) are associated with autonomic dysregulation, including elevated sympathetic nervous system activity. Altered nucleus tractus solitarii (nTS) activity contributes to this response. Neurons originating in the paraventricular nucleus (PVN), including those containing oxytocin (OT) and corticotropin-releasing hormone (CRH), project to the nTS, and modulate the cardiorespiratory system. Their role in CIH is unknown. In this study, we focused on OT and CRH individually and together on nTS activity from rats exposed to either CIH or normoxia control. We show that after CIH, CRH alone and with OT increased to a greater extent overall nTS discharge, neuronal calcium influx, synaptic transmission to second-order nTS neurons, and OT and CRH receptor expression. These results provide insights into the underlying circuits and mechanisms contributing to autonomic dysfunction during periods of episodic breathing.

Kv2 channels contribute to neuronal activity within the vagal afferent-nTS reflex arc.

Diversity in the functional expression of ion channels contributes to the unique patterns of activity generated in visceral sensory A-type myelinated neurons versus C-type unmyelinated neurons in response to their natural stimuli. In the present study, Kv2 channels were identified as underlying a previously uncharacterized delayed rectifying potassium current expressed in both A- and C-type nodose ganglion neurons. Kv2.1 and 2.2 appear confined to the soma and initial segment of these sensory neurons; however, neither was identified in their central presynaptic terminals projecting onto relay neurons in the nucleus of the solitary tract (nTS). Kv2.1 and Kv2.2 were also not detected in the peripheral axons and sensory terminals in the aortic arch. Functionally, in nodose neuron somas, Kv2 currents exhibited frequency-dependent current inactivation and contributed to action potential repolarization in C-type neurons but not A-type neurons. Within the nTS, the block of Kv2 currents does not influence afferent presynaptic calcium influx or glutamate release in response to afferent activation, supporting our immunohistochemical observations. On the other hand, Kv2 channels contribute to membrane hyperpolarization and limit action potential discharge rate in second-order neurons. Together, these data demonstrate that Kv2 channels influence neuronal discharge within the vagal afferent-nTS circuit and indicate they may play a significant role in viscerosensory reflex function.NEW & NOTEWORTHY We demonstrate the expression and function of the voltage-gated delayed rectifier potassium channel Kv2 in vagal nodose neurons. Within sensory neurons, Kv2 channels limit the width of the broader C-type but not narrow A-type action potential. Within the nucleus of the solitary tract (nTS), the location of the vagal terminal field, Kv2 does not influence glutamate release. However, Kv2 limits the action potential discharge of nTS relay neurons. These data suggest a critical role for Kv2 in the vagal-nTS reflex arc.

Hypoxia augments TRPM3-mediated calcium influx in vagal sensory neurons.

Transient receptor potential melastatin 3 (TRPM3) channels contribute to nodose afferent and brainstem nucleus tractus solitarii (nTS) activity. Exposure to short, sustained hypoxia (SH) and chronic intermittent hypoxia (CIH) enhances nTS activity, although the mechanisms are unknown. We hypothesized TRPM3 may contribute to increased neuronal activity in nTS-projecting nodose ganglia viscerosensory neurons, and its influence is elevated following hypoxia. Rats were exposed to either room air (normoxia), 24-h of 10 % O2 (SH), or CIH (episodic 6 % O2 for 10d). A subset of neurons from normoxic rats were exposed to in vitro incubation for 24-h in 21 % or 1 % O2. Intracellular Ca2+ of dissociated neurons was monitored via Fura-2 imaging. Ca2+ levels increased upon TRPM3 activation via Pregnenolone sulfate (Preg) or CIM0216. Preg responses were eliminated by the TRPM3 antagonist ononetin, confirming agonist specificity. Removal of extracellular Ca2+ also eliminated Preg response, further suggesting Ca2+ influx via membrane-bound channels. In neurons isolated from SH-exposed rats, the TRPM3 elevation of Ca2+ was greater than in normoxic-exposed rats. The SH increase was reversed following a subsequent normoxic exposure. RNAScope demonstrated TRPM3 mRNA was greater after SH than in Norm ganglia. Incubating dissociated cultures from normoxic rats in 1 % O2 (24-h) did not alter the Preg Ca2+ responses compared to their normoxic controls. In contrast to in vivo SH, 10d CIH did not alter TRPM3 elevation of Ca2+. Altogether, these results demonstrate a hypoxia-specific increase in TRPM3-mediated calcium influx.

Image reconstruction algorithm for laser-induced ultrasonic imaging: The single sensor scanning synthetic aperture focusing technique.

This paper aims to implement a laser-induced ultrasound imaging reconstruction method based on the delay-and-sum beamforming through the synthetic aperture focusing technique (SAFT) for a circular scanning, performed with a tomograph that had one acoustic sensor and a system that rotates the sample around a fixed axis. The proposed method, called the Single-sensor Scanning Synthetic Aperture Focusing Technique, considers the size of the sensor and the detection procedure inside the SAFT's algebra. This image reconstruction method was evaluated numerically, using the Green function for the laser-induced ultrasound wave equation to generate a forward problem, and experimentally, using a solid object of polylactic acid, and a Sprague-Dawley rat heart located in a tissue-mimicking phantom. The resulting images were compared to those obtained from the time reversal and the conventional delay-and-sum reconstruction algorithms. The presented method removes the sidelobe artifacts and the comet tail sign, which produces a more distinguishable target on the image. In addition, the proposed method has a faster performance and lower computational load. The implementation of this method in photoacoustic microscopy techniques for image reconstruction is discussed.

Exaggerated potassium current reduction by oxytocin in visceral sensory neurons following chronic intermittent hypoxia.

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and systemically in response to a variety of stimuli and stressors, including hypoxia. Within the nucleus tractus solitarii (nTS), the first integration site for cardiorespiratory reflexes, OT enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. The nTS receives sensory input from baroafferent neurons that originate in the nodose ganglia. Nodose neurons express the OT receptor (OTR) whose activation elevates intracellular calcium. However, the influence of OT on other ion channels, especially potassium channels important for neuronal activity during CIH, is less known. This study sought to determine the mechanism (s) by which OT modulates sensory afferent-nTS mediated reflexes normally and after CIH. Nodose ganglia neurons from male Sprague-Dawley rats were examined after 10d CIH (6% O2 every 3 min) or their normoxic (21% O2) control. OTR mRNA and protein were identified in Norm and CIH ganglia and was similar between groups. To examine OTR function, APs and potassium currents (IK) were recorded in dissociated neurons. Compared to Norm, after CIH OT depolarized neurons and reduced current-induced AP discharge. After CIH OT also produced a greater reduction in IK that where tetraethylammonium-sensitive. These data demonstrate after CIH OT alters ionic currents in nodose ganglia cells to likely influence cardiorespiratory reflexes and overall function.

Activation of alpha-1 adrenergic receptors increases cytosolic calcium in neurones of the paraventricular nucleus of the hypothalamus.

Norepinephrine (NE) activates adrenergic receptors (ARs) in the hypothalamic paraventricular nucleus (PVN) to increase excitatory currents, depolarise neurones and, ultimately, augment neuro-sympathetic and endocrine output. Such cellular events are known to potentiate intracellular calcium ([Ca2+ ]i ); however, the role of NE with respect to modulating [Ca2+ ]i in PVN neurones and the mechanisms by which this may occur remain unclear. We evaluated the effects of NE on [Ca2+ ]i of acutely isolated PVN neurones using Fura-2 imaging. NE induced a slow increase in [Ca2+ ]i compared to artificial cerebrospinal fluid vehicle. NE-induced Ca2+ elevations were mimicked by the α1 -AR agonist phenylephrine (PE) but not by α2 -AR agonist clonidine (CLON). NE and PE but not CLON also increased the overall number of neurones that increase [Ca2+ ]i (ie, responders). Elimination of extracellular Ca2+ or intracellular endoplasmic reticulum Ca2+ stores abolished the increase in [Ca2+ ]i and reduced responders. Blockade of voltage-dependent Ca2+ channels abolished the α1 -AR induced increase in [Ca2+ ]i and number of responders, as did inhibition of phospholipase C inhibitor, protein kinase C and inositol triphosphate receptors. Spontaneous phasic Ca2+ events, however, were not altered by NE, PE or CLON. Repeated K+ -induced membrane depolarisation produced repetitive [Ca2+ ]i elevations. NE and PE increased baseline Ca2+ , whereas NE decreased the peak amplitude. CLON also decreased peak amplitude but did not affect baseline [Ca2+ ]i . Taken together, these data suggest receptor-specific influence of α1 and α2 receptors on the various modes of calcium entry in PVN neurones. They further suggest Ca2+ increase via α1 -ARs is co-dependent on extracellular Ca2+ influx and intracellular Ca2+ release, possibly via a phospholipase C inhibitor-mediated signalling cascade.

Hydrogen peroxide inhibits neurons in the paraventricular nucleus of the hypothalamus via potassium channel activation.

The paraventricular nucleus (PVN) of the hypothalamus is an important homeostatic and reflex center for neuroendocrine, respiratory, and autonomic regulation, including during hypoxic stressor challenges. Such challenges increase reactive oxygen species (ROS) to modulate synaptic, neuronal, and ion channel activity. Previously, in the nucleus tractus solitarius, another cardiorespiratory nucleus, we showed that the ROS H2O2 induced membrane hyperpolarization and reduced action potential discharge via increased K+ conductance at the resting potential. Here, we sought to determine the homogeneity of influence and mechanism of action of H2O2 on PVN neurons. We recorded PVN neurons in isolation and in an acute slice preparation, which leaves neurons in their semi-intact network. Regardless of preparation, H2O2 hyperpolarized PVN neurons and decreased action potential discharge. In the slice preparation, H2O2 also decreased spontaneous excitatory postsynaptic current frequency, but not amplitude. To examine potential mechanisms, we investigated the influence of the K+ channel blockers Ba2+, Cs+, and glibenclamide on membrane potential, as well as the ionic currents active at resting potential and outward K+ currents (IK) upon depolarization. The H2O2 hyperpolarization was blocked by K+ channel blockers. H2O2 did not alter currents between -50 and -110 mV. However, H2O2 induced an outward IK at -50 mV yet, at potentials more positive to 0 mV H2O2, decreased IK. Elevated intracellular antioxidant catalase eliminated H2O2 effects. These data indicate that H2O2 alters synaptic and neuronal properties of PVN neurons likely via membrane hyperpolarization and alteration of IK, which may ultimately alter cardiorespiratory reflexes.

H2O2 augments cytosolic calcium in nucleus tractus solitarii neurons via multiple voltage-gated calcium channels.

Reactive oxygen species (ROS) play a profound role in cardiorespiratory function under normal physiological conditions and disease states. ROS can influence neuronal activity by altering various ion channels and transporters. Within the nucleus tractus solitarii (nTS), a vital brainstem area for cardiorespiratory control, hydrogen peroxide (H2O2) induces sustained hyperexcitability following an initial depression of neuronal activity. The mechanism(s) associated with the delayed hyperexcitability are unknown. Here we evaluate the effect(s) of H2O2 on cytosolic Ca2+ (via fura-2 imaging) and voltage-dependent calcium currents in dissociated rat nTS neurons. H2O2 perfusion (200 µM; 1 min) induced a delayed, slow, and moderate increase (~27%) in intracellular Ca2+ concentration ([Ca2+]i). The H2O2-mediated increase in [Ca2+]i prevailed during thapsigargin, excluding the endoplasmic reticulum as a Ca2+ source. The effect, however, was abolished by removal of extracellular Ca2+ or the addition of cadmium to the bath solution, suggesting voltage-gated Ca2+ channels (VGCCs) as targets for H2O2 modulation. Recording of the total voltage-dependent Ca2+ current confirmed H2O2 enhanced Ca2+ entry. Blocking VGCC L, N, and P/Q subtypes decreased the number of cells and their calcium currents that respond to H2O2 The number of responder cells to H2O2 also decreased in the presence of dithiothreitol, suggesting the actions of H2O2 were dependent on sulfhydryl oxidation. In summary, here, we have shown that H2O2 increases [Ca2+]i and its Ca2+ currents, which is dependent on multiple VGCCs likely by oxidation of sulfhydryl groups. These processes presumably contribute to the previously observed delayed hyperexcitability of nTS neurons in in vitro brainstem slices.

5-hydroxytryptamine 2C receptors tonically augment synaptic currents in the nucleus tractus solitarii.

The nucleus tractus solitarii (nTS) is the primary termination and integration point for visceral afferents in the brain stem. Afferent glutamate release and its efficacy on postsynaptic activity within this nucleus are modulated by additional neuromodulators and transmitters, including serotonin (5-HT) acting through its receptors. The 5-HT(2) receptors in the medulla modulate the cardiorespiratory system and autonomic reflexes, but the distribution of the 5-HT(2C) receptor and the role of these receptors during synaptic transmission in the nTS remain largely unknown. In the present study, we examined the distribution of 5-HT(2C) receptors in the nTS and their role in modulating excitatory postsynaptic currents (EPSCs) in monosynaptic nTS neurons in the horizontal brain stem slice. Real-time RT-PCR and immunohistochemistry identified 5-HT(2C) receptor message and protein in the nTS and suggested postsynaptic localization. In nTS neurons innervated by general visceral afferents, 5-HT(2C) receptor activation increased solitary tract (TS)-EPSC amplitude and input resistance and depolarized membrane potential. Conversely, 5-HT(2C) receptor blockade reduced TS-EPSC and miniature EPSC amplitude, as well as input resistance, and hyperpolarized membrane potential. Synaptic parameters in nTS neurons that receive sensory input from carotid body chemoafferents were also attenuated by 5-HT(2C) receptor blockade. Taken together, these data suggest that 5-HT(2C) receptors in the nTS are located postsynaptically and augment excitatory neurotransmission.

Hydrogen sulfide augments synaptic neurotransmission in the nucleus of the solitary tract.

Within the brain stem, the nucleus tractus solitarii (NTS) serves as a principal central site for sensory afferent integration from the cardiovascular and respiratory reflexes. Neuronal activity and synaptic transmission in the NTS are highly pliable and subject to neuromodulation. In the central nervous system, hydrogen sulfide (H2S) is a gasotransmitter generated primarily by the enzyme cystathionine-ß-synthase (CBS). We sought to determine the role of H2S, and its generation by CBS, in NTS excitability. Real-time RT-PCR, immunoblot, and immunohistochemistry analysis identified the presence of CBS in the NTS. Patch-clamp electrophysiology in brain stem slices examined excitatory postsynaptic currents (EPSCs) and membrane properties in monosynaptically driven NTS neurons. Confocal imaging of labeled afferent synaptic terminals in NTS slices monitored intracellular calcium. Exogenous H2S significantly increased the amplitude of evoked solitary tract (TS)-EPSCs, frequency of miniature (m)EPSCs, and presynaptic terminal calcium fluorescence in the NTS. H2S did not alter action potential discharge or postsynaptic properties. On the other hand, the CBS inhibitor aminooxyacetate (AOA) significantly reduced the amplitude of TS-EPSCs and presynaptic terminal calcium fluorescence in the NTS without altering postsynaptic properties. Taken together, these data support a presynaptic role for endogenous H2S in modulation of excitatory neurotransmission in the NTS.

Age affects spontaneous activity and depolarizing afterpotentials in isolated gonadotropin-releasing hormone neurons.

Neuronal activity underlying the pulsatile secretion of GnRH remains poorly understood, as does the endogenous generation of such activity. It is clear that changes at the level of the hypothalamus are taking place during reproductive aging, yet virtually nothing is known about GnRH neuronal physiology in aging and postreproductive animals. In these studies, we performed cell-attached and whole-cell recordings in GnRH-enhanced green fluorescent protein neurons dissociated from young (3 months), middle-aged (10 months), and old (15-18 months) female mice. All mice were ovariectomized; half were estradiol replaced. Neurons from all ages fired spontaneously, most in a short-burst pattern that is characteristic of GnRH neuronal firing. Membrane characteristics were not affected by age. However, firing frequency was significantly reduced in neurons from old animals, as was spike patterning. The amplitude of the depolarizing afterpotential, evoked by a 200-msec current pulse, was significantly smaller in aged animals. In addition, inward whole-cell currents were reduced in estradiol-treated animals, although they were not significantly affected by age. Because depolarizing afterpotentials have been shown to contribute to prolonged discharges of activity after a very brief excitatory input, a decreased depolarizing afterpotential could lead to attenuated pulses in older animals. In addition, decreases in frequency and pattern generation could lead to improper information coding. Therefore, changes in the GnRH neuron during aging could lead to dysregulated activity, potentially resulting in the attenuated LH pulses observed in the transition to reproductive senescence.