David D. Kline

Peripheral cardiorespiratory reflexes respond to various stressors and are processed in the brainstem nucleus tractus solitarii (nTS). The paraventricular nucleus of the hypothalamus is reciprocally connected to the nTS and influences cardiorespiratory function partly through corticotropin-releasing hormone (CRH) projections to the nTS. Our previous studies showed that CRH enhances nTS activity and increases cardiorespiratory responses to hypoxia. However, the specific effects of CRH on the unique nTS neuron phenotypes remain unclear. We hypothesized that CRH's general excitatory effect on nTS function results from its differential influence on nTS phenotypes. To test this hypothesis, we examined the electrophysiological and dynamic calcium fluorescent responses to CRH, as well as the CRH receptor profiles, of GABA neurons within the nTS of Gad1-EGFP mice (male and female) and compared them with non-GABA neurons. CRH-positive synaptic terminals were adjacent to both neuron phenotypes. The expression of the CRH receptor, crhr1 , was greater in GABA versus non-GABA neurons in the medial nTS. Functionally, CRH reduced afferent (TS)-evoked EPSC amplitudes in GABA neurons but increased current amplitude in non-GABA neurons. Applying a CRH receptor blocker before CRH abolished synaptic responses in both groups, confirming the specificity of CRH action. GABA neuron synaptic activity was tonically restrained by CRH, as CRH receptor blockade increased TS-EPSC amplitude. Our findings show that CRH effects on nTS activity differ by neuron type, with GABA neuron activity inhibited by CRH. Such responses likely contribute to CRH's excitatory influence in the nTS and to the elevated breathing and sympathoexcitation observed during hypoxia.

Cardiovascular and respiratory responses to sustained hypoxia (SH) in rats and mice are different. To understand the contribution of changes in synaptic transmission in nucleus tractus solitarius (NTS) neurons of C57Bl/6J mice to their responses to SH we evaluated the following: (1) the neuronal excitability and excitatory synaptic transmission in the NTS neurons, (2) in what level of the synapses (pre‐ and postsynaptic) and also(3) to what extent neuron‐astrocyte interaction contribute to these changes. Electrophysiological, immunohistochemical and intracellular Ca ²⁺ imaging approaches in NTS slices after normoxia (FIO _2 =  0.21, 24 h) or SH (FIO _2 =  0.10, 24 h) were used. SH increased AMPA and NMDA currents in NTS neurons in response to solitary tract (TS) stimulation, indicating increased glutamatergic excitatory transmission. The number of action potentials after injection of positive current and TS stimulation (10 Hz) was increased by SH. Spontaneous extracellular activity in the NTS also increased, suggesting increased neuronal network activity after SH. The presynaptic mechanisms and the neuron‐astrocyte interaction were not affected, but SH increased the amplitude of postsynaptic currents in NTS neurons induced by AMPA and NMDA perfusion. Therefore the enhancement of neuronal excitability and excitatory synaptic transmission in the NTS neurons of mice in response to SH is due to postsynaptic changes rather than changes in presynaptic parameters or in neuron‐astrocyte interaction. The findings that the mechanisms underlying the increase in the excitatory synaptic transmission in the NTS of mice and rats are not the same contribute to explain the distinct cardiovascular and respiratory adjustments in mice and rats when exposed to SH. image Key points: Cardiovascular and respiratory responses to sustained hypoxia (SH) in rats and mice are different. The extent to which changes in synaptic transmission in nucleus tractus solitarius (NTS) contribute to these responses is unclear. SH increases excitatory postsynaptic currents in NTS neurons of mice in response to solitary tract (TS) stimulation or to AMPA and NMDA perfusion. SH increases the number of action potentials in NTS neurons in response to injected current and spontaneous extracellular activity evaluated by a multielectrode array. SH did not alter presynaptic parameters, neuron‐astrocyte interaction or morphological and electrophysiological properties of astrocytes. Our data show that the overall mechanisms underlying changes in the excitatory synaptic transmission in the NTS neurons of mice in response to SH are different in relation to rats. Although in rats it was documented changes in astrocytic modulation and postsynaptic currents, in mice we are showing that it is restricted to changes in postsynaptic currents.

Exposure to acute intermittent hypoxia (AIH) induced sustained increases in phrenic nerve activity (phrenic long-term facilitation, pLTF) that were associated with increased nucleus tractus solitarii (nTS) neuronal Ca ²⁺ and action potential discharge. nTS CaMKII activity was critical for pLTF development and AIH-induced increases in neuronal discharge. In contrast, nTS NMDA receptor (NMDAR) activation, but not CaMKII activity, was required for maintaining AIH-induced pLTF. Increased nTS neuronal activity, NMDAR activation, and CaMKII are critical for AIH-induced neuroplasticity in central cardiorespiratory output.

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. image 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.

Viscerosensory information travels to the brain via vagal afferents, where it is first integrated within the brainstem nucleus tractus solitarii (nTS), a critical contributor to cardiorespiratory function and site of neuroplasticity. We have shown that decreasing input to the nTS via unilateral vagus nerve transection (vagotomy) induces morphological changes in nTS glia and reduces sighs during hypoxia. The mechanisms behind post‐vagotomy changes are not well understood. We hypothesized that chronic vagotomy alters cardiorespiratory responses to vagal afferent stimulation via blunted nTS neuronal activity. Male Sprague–Dawley rats (6 weeks old) underwent right cervical vagotomy caudal to the nodose ganglion, or sham surgery. After 1 week, rats were anaesthetized, ventilated and instrumented to measure mean arterial pressure (MAP), heart rate (HR), and splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA, respectively). Vagal afferent stimulation (2–50 Hz) decreased cardiorespiratory parameters and increased neuronal Ca²⁺ measured by in vivo photometry and in vitro slice imaging of nTS GCaMP8m. Vagotomy attenuated both these reflex and neuronal Ca²⁺ responses compared to shams. Vagotomy also reduced presynaptic Ca²⁺ responses to stimulation (Cal‐520 imaging) in the nTS slice. The decrease in HR, SSNA and PhrNA due to nTS nanoinjection of exogenous glutamate also was tempered following vagotomy. This effect was not restored by blocking excitatory amino acid transporters. However, the blunted responses were mimicked by NMDA, not AMPA, nanoinjection and were associated with reduced NR1 subunits in the nTS. Altogether, these results demonstrate that vagotomy induces multiple changes within the nTS tripartite synapse that influence cardiorespiratory reflex responses to afferent stimulation. image Key points: Multiple mechanisms within the nucleus tractus solitarii (nTS) contribute to functional changes following vagal nerve transection. Vagotomy results in reduced cardiorespiratory reflex responses to vagal afferent stimulation and nTS glutamate nanoinjection. Blunted responses occur via reduced presynaptic Ca²⁺ activation and attenuated NMDA receptor expression and function, leading to a reduction in nTS neuronal activation. These results provide insight into the control of autonomic and respiratory function, as well as the plasticity that can occur in response to nerve damage and cardiorespiratory disease.

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.

The hypothalamic paraventricular nucleus (PVN) is essential to peripheral chemoreflex neurocircuitry, but the specific efferent pathways utilized are not well defined. The PVN sends dense projections to the nucleus tractus solitarii (nTS), which exhibits neuronal activation following a hypoxic challenge. We hypothesized that nTS‐projecting PVN (PVN‐nTS) neurons contribute to hypoxia‐induced nTS neuronal activation and cardiorespiratory responses. To selectively target PVN‐nTS neurons, rats underwent bilateral nTS nanoinjection of retrogradely transported adeno‐associated virus (AAV) driving Cre recombinase expression. We then nanoinjected into PVN AAVs driving Cre‐dependent expression of Gq or Gi designer receptors exclusively activated by designer drugs (DREADDs) to test the degree that selective activation or inhibition, respectively, of the PVN‐nTS pathway affects the hypoxic ventilatory response (HVR) of conscious rats. We used immunohistochemistry for Fos and extracellular recordings to examine how DREADD activation influences PVN‐nTS neuronal activation by hypoxia. Pathway activation enhanced the HVR at moderate hypoxic intensities and increased PVN and nTS Fos immunoreactivity in normoxia and hypoxia. In contrast, PVN‐nTS inhibition reduced both the HVR and PVN and nTS neuronal activation following hypoxia. To further confirm selective pathway effects on central cardiorespiratory output, rats underwent hypoxia before and after bilateral nTS nanoinjections of C21 to activate or inhibit PVN‐nTS terminals. PVN terminal activation within the nTS enhanced tachycardic, sympathetic and phrenic (PhrNA) nerve activity responses to hypoxia whereas inhibition attenuated hypoxia‐induced increases in nTS neuronal action potential discharge and PhrNA. The results demonstrate the PVN‐nTS pathway enhances nTS neuronal activation and is necessary for full cardiorespiratory responses to hypoxia. image Key points: The hypothalamic paraventricular nucleus (PVN) contributes to peripheral chemoreflex cardiorespiratory responses, but specific PVN efferent pathways are not known. The nucleus tractus solitarii (nTS) is the first integration site of the peripheral chemoreflex, and the nTS receives dense projections from the PVN. Selective GqDREADD activation of the PVN‐nTS pathway was shown to enhance ventilatory responses to hypoxia and activation (Fos immunoreactivity (IR)) of nTS neurons in conscious rats, augmenting the sympathetic and phrenic nerve activity (SSNA and PhrNA) responses to hypoxia in anaesthetized rats. Selective GiDREADD inhibition of PVN‐nTS neurons attenuates ventilatory responses, nTS neuronal Fos‐IR, action potential discharge and PhrNA responses to hypoxia. These results demonstrate that a projection from the PVN to the nTS is critical for full chemoreflex responses to hypoxia.

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.