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Objective Severe periictal respiratory depression is thought to be linked to risk of sudden unexpected death in epilepsy (SUDEP) but its determinants are largely unknown. Interindividual differences in the interictal ventilatory response to CO2 (hypercapnic ventilatory response [HCVR] or central respiratory CO2 chemosensitivity) may identify patients who are at increased risk for severe periictal hypoventilation. HCVR has not been studied previously in patients with epilepsy; therefore we evaluated a method to measure it at bedside in an epilepsy monitoring unit (EMU) and examined its relationship to postictal hypercapnia following generalized convulsive seizures (GCSs). Methods Interictal HCVR was measured by a respiratory gas analyzer using a modified rebreathing technique. Minute ventilation (VE ), tidal volume, respiratory rate, end tidal (ET) CO2 and O2 were recorded continuously. Dyspnea during the test was assessed using a validated scale. The HCVR slope (ΔVE /ΔETCO2 ) for each subject was determined by linear regression. During the video-electroencephalography (EEG) study, subjects underwent continuous respiratory monitoring, including measurement of chest and abdominal movement, oronasal airflow, transcutaneous (tc) CO2 , and capillary oxygen saturation (SPO2 ). Results Sixty-eight subjects completed HCVR testing in 151 ± (standard deviation) 58 seconds, without any serious adverse events. HCVR slope ranged from -0.94 to 5.39 (median 1.71) L/min/mm Hg. HCVR slope correlated with the degree of unpleasantness and intensity of dyspnea and was inversely related to baseline ETCO2 . Both the duration and magnitude of postictal tcCO2 rise following GCSs were inversely correlated with HCVR slope. Significance Measurement of the HCVR is well tolerated and can be performed rapidly and safely at the bedside in the EMU. A subset of individuals has a very low sensitivity to CO2 , and this group is more likely to have a prolonged increase in postictal CO2 after GCS. Low interictal HCVR may increase the risk of severe respiratory depression and SUDEP after GCS and warrants further study.

OBJECTIVE: Central CO(2) chemoreception (CCR), a major chemical drive for breathing, can be quantified with a CO(2) re-breathing test to measure the hypercapnic ventilatory response (HCVR). An attenuated HCVR correlates with the severity of respiratory dysfunction after generalized convulsive seizures and is a potential biomarker for sudden unexpected death in epilepsy (SUDEP) risk. Vagus nerve stimulation (VNS) may reduce SUDEP risk, but for unclear reasons the risk remains higher during the first 2 years after implantation. The vagus nerve has widespread connections in the brainstem, including key areas related to CCR. Here we examined whether chronic electrical stimulation of the vagus nerve induces changes in CCR. METHODS: We compared the HCVR in epilepsy patients with or without an active VNS in a sex- and age-matched case-control study. Eligible subjects were selected from a cohort of patients who previously underwent HCVR testing. The HCVR slope, change in minute ventilation (VE) with respect to change in end-tidal (ET) CO(2) (Δ VE/ Δ ETCO(2)) during the test was calculated for each subject. Key variables were compared between the two groups. Univariate and multivariate analyses were carried out for HCVR slope as dependent variable. RESULTS: A total of 86 subjects were in the study. HCVR slope was significantly lower in the cases compared to the controls. Cases had longer duration of epilepsy and higher number of anti-epileptic drugs (AEDs) tried during lifetime. Having active VNS and ETCO(2) were associated with a low HCVR slope while high BMI was associated with high HCVR slope in both univariate and multivariate analyses. DISCUSSION: We found having an active VNS was associated with relatively attenuated HCVR slope. Although duration of epilepsy and number of AEDs tried during lifetime was significantly different between the groups, they were not predictors of HCVR slope in subsequent analysis. CONCLUSION: Chronic electrical stimulation of the vagus nerve by VNS may be associated with an attenuated CCR. A larger prospective study may help to establish the time course of this effect in relation to the time of VNS implantation, whether there is a causal relationship, and determine how it affects SUDEP risk.

Background Recent reports of fatal or near-fatal events in epilepsy monitoring units (EMUs) and an increasing awareness of the effects of seizures on breathing have stimulated interest in cardiorespiratory monitoring for patients undergoing video-electroencephalography (EEG) recording. Patient and provider acceptance of these extra recording devices has not previously been studied and may represent a barrier to widespread adoption. Methods We queried EMU subjects regarding their experiences with a monitoring protocol that included the continuous measurement of oral/nasal airflow, respiratory effort (chest and abdominal respiratory inductance plethysmography), oxygen saturation, and transcutaneous CO2. Surveys were returned by 71.4% (100/140) of eligible subjects. Results Overall, 73% of participants reported being moderately to highly satisfied with the monitoring, and 82% reported moderate to strong agreement that advance knowledge of the monitoring would not have changed their decision to proceed with the video-EEG study. Except for nasal airflow, none of the additional monitoring devices caused more discomfort than EEG electrodes. Conclusion Patient acceptance of an EMU comprehensive cardiorespiratory monitoring protocol is high. The information obtained from “multimodality recording” should help clinicians and investigators understand the effect of seizures on both cardiac and respiratory physiology, may enhance safety in the EMU, and may aid in the identification of biomarkers for sudden unexpected death in epilepsy (SUDEP).

Author(s): Lacuey, Nuria; Zonjy, Bilal; Hampson, Johnson P; Rani, MR Sandhya; Zaremba, Anita; Sainju, Rup K; Gehlbach, Brian K; Schuele, Stephan; Friedman, Daniel; Devinsky, Orrin; Nei, Maromi; Harper, Ronald M; Allen, Luke; Diehl, Beate; Millichap, John J; Bateman, Lisa; Granner, Mark A; Dragon, Deidre N; Richerson, George B; Lhatoo, Samden D | Abstract: OBJECTIVE:The aim of this study was to investigate periictal central apnea as a seizure semiological feature, its localizing value, and possible relationship with sudden unexpected death in epilepsy (SUDEP) pathomechanisms. METHODS:We prospectively studied polygraphic physiological responses, including inductance plethysmography, peripheral capillary oxygen saturation (SpO2 ), electrocardiography, and video electroencephalography (VEEG) in 473 patients in a multicenter study of SUDEP. Seizures were classified according to the International League Against Epilepsy (ILAE) 2017 seizure classification based on the most prominent clinical signs during VEEG. The putative epileptogenic zone was defined based on clinical history, seizure semiology, neuroimaging, and EEG. RESULTS:Complete datasets were available in 126 patients in 312 seizures. Ictal central apnea (ICA) occurred exclusively in focal epilepsy (51/109 patients [47%] and 103/312 seizures [36.5%]) (P l .001). ICA was the only clinical manifestation in 16/103 (16.5%) seizures, and preceded EEG seizure onset by 8 ± 4.9 s, in 56/103 (54.3%) seizures. ICA ≥60 s was associated with severe hypoxemia (SpO2 l75%). Focal onset impaired awareness (FOIA) motor onset with automatisms and FOA nonmotor onset semiologies were associated with ICA presence (P l .001), ICA duration (P = .002), and moderate/severe hypoxemia (P = .04). Temporal lobe epilepsy was highly associated with ICA in comparison to extratemporal epilepsy (P = .001) and frontal lobe epilepsy (P = .001). Isolated postictal central apnea was not seen; in 3/103 seizures (3%), ICA persisted into the postictal period. SIGNIFICANCE:ICA is a frequent, self-limiting semiological feature of focal epilepsy, often starting before surface EEG onset, and may be the only clinical manifestation of focal seizures. However, prolonged ICA (≥60 s) is associated with severe hypoxemia and may be a potential SUDEP biomarker. ICA is more frequently seen in temporal than extratemporal seizures, and in typical temporal seizure semiologies. ICA rarely persists after seizure end. ICA agnosia is typical, and thus it may remain unrecognized without polygraphic measurements that include breathing parameters.

Saporin (SAP) or SAP conjugates injected into the nucleus tractus solitarii (NTS) of rats kill astrocytes. When injected in its unconjugated form, SAP produces no demonstrable loss of or damage to local neurons. However bilateral injections of SAP significantly attenuate responses to activation of baroreceptor reflexes that are mediated by transmission of signals through glutamate receptors in the NTS. We tested the hypothesis that SAP would reduce cardiovascular responses to activation of NTS glutamate receptors despite its recognized ability to spare local neurons while killing local astrocytes. In animals treated with SAP and SAP conjugates or, as a control, with the toxin 6‐hydroxydopamine (6‐OHDA), we sought to determine if dose‐related changes of arterial pressure (AP) or heart rate (HR) in response to injection into NTS of N‐methyl‐d‐aspartate (NMDA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) were attenuated. Also we quantified changes in immunoreactivity (IR) for EAAT2, EAAC1, and VGluT2 in NTS after SAP and SAP conjugates. Our earlier studies showed that IR for NMDA and AMPA receptors was not changed after injection of SAP. We found that EAAT2 and EAAC1, both found in astrocytes, were reduced by SAP or its conjugates but not by 6‐OHDA. In contrast, VGluT2‐IR was increased by SAP or conjugates but not by 6‐OHDA. AP and HR responses to NMDA and AMPA were attenuated after SAP and SAP conjugate injection but not after 6‐OHDA. Results of this study are consistent with others that have shown interactions between astroglia and neurons in synaptic transmission mediated by glutamate receptor activation in the NTS.

Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex.

Injection into the nucleus tractus solitarii (NTS) of toxins that target substance P (SP) receptors ablates neurons that express neurokinin-1 (NK1) receptors, attenuates baroreflexes, and results in increased lability of arterial pressure. We and others have shown that the toxin leads to loss of neurons containing SP receptors and loss of GABAergic neurons in the NTS; but given that neither type neuron is thought to be integral to baroreflex transmission in NTS, mechanisms responsible for the cardiovascular changes remained unclear. Because NK1 receptors colocalize with N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in NTS and because glutamate transmission may be integral to baroreflex transmission in the NTS we hypothesized that the toxic lesions may interrupt mechanisms for glutamate transmission. Interruption of those mechanisms could be responsible for the cardiovascular effects. We tested the hypothesis by performing fluorescent immunohistochemistry, confocal microscopy and image analysis after injecting stabilized SP-SAP (SSP-SAP) unilaterally into the NTS. We assessed changes in immunoreactivity (IR) of NMDA receptor subunit 1 (NMDAR1), AMPA receptor subunit 2 (GluR2), and 3 types of vesicular glutamate transporters (VGluT) as well as IR of gamma-aminobutyric acid receptors type b (GABAb), neuronal nitirc oxide synthase (nNOS), tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP 9.5), a neuronal marker, in the NTS. When compared to that of the same section of the un-injected NTS, IR decreased significantly in the injected side for NMDAR1 (p < 0.01), GluR2 (p < 0.01), VGluT3 (p < 0.01), GABAb (p < 0.001), and PGP9.5 (p < 0.001). In contrast, IR for VGluT1 (p < 0.001), VGluT2 (p < 0.001), nNOS (p < 0.001), and TH (p < 0.001) increased significantly. We conclude that pathologic effects following ablation of neurons with NK1 receptors in NTS may result from interruption of neurotransmission through other neurochemical systems associated with NK1 receptors-containing neurons.

Adeno-associated virus (AAV) has distinct advantages over other viral vectors in delivering genes of interest to the brain. AAV mainly transfects neurons, produces no toxicity or inflammatory responses, and yields long-term transgene expression. In this study, we first tested the hypothesis that AAV serotype 2 (AAV2) selectively transfects neurons but not glial cells in the nucleus tractus solitarii (NTS) by examining expression of the reporter gene, enhanced green fluorescent protein (eGFP), in the rat NTS after unilateral microinjection of AAV2eGFP into NTS. Expression of eGFP was observed in 1–2 cells in the NTS 1 day after injection. The number of transduced cells and the intensity of eGFP fluorescence increased from day 1 to day 28 and decreased on day 60. The majority (92.9 ± 7.0%) of eGFP expressing NTS cells contained immunoreactivity for the neuronal marker, protein gene product 9.5, but not that for the glial marker, glial fibrillary acidic protein. We observed eGFP expressing neurons and fibers in the nodose ganglia (NG) both ipsilateral and contralateral to the injection. In addition, eGFP expressing fibers were present in both ipsilateral and contralateral nucleus ambiguus (NA), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). Having established that AAV2 was able to transduce a gene into NTS neurons, we constructed AAV2 vectors that contained cDNA for neuronal nitric oxide synthase (nNOS) and examined nNOS expression in the rat NTS after injection of this vector into the area. Results from RT-PCR, Western analysis, and immunofluorescent histochemistry indicated that nNOS expression was elevated in rat NTS that had been injected with AAV2nNOS vectors. Therefore, we conclude that AAV2 is an effective viral vector in chronically transducing NTS neurons and that AAV2nNOS can be used as a specific gene transfer tool to study the role of nNOS in CNS neurons.

Parasympathetic nerves from the pterygopalatine ganglia may participate in development of cluster headaches, in vascular responses to hypertension and in modulation of damage due to stroke. Stimulation of the nerves elicits cerebral vasodilatation, but it is not known if the nerves tonically influence cerebrovascular tone. We hypothesized that parasympathetics provide a tonic vasodilator influence and tested that hypothesis by measuring cerebral blood flow in anesthetized rats before and after removal of a pterygopalatine ganglion. Ganglion removal led to reduced cerebral blood flow without changing blood pressure. Thus, parasympathetic nerves provide tonic vasodilatory input to cerebral blood vessels.

Forebrain arteries receive nitroxidergic input from parasympathetic ganglionic fibers that arise from the pterygopalatine ganglia. Previous studies have shown that ganglionic stimulation in some species led to cerebral vasodilatation while interruption of those fibers interfered with vasodilatation seen during acute hypertension. Because the ganglionic fibers are quite delicate and are easily damaged when the ganglia are approached with published techniques we sought to develop a method that allowed clear exposure of the ganglia and permitted demonstration of cerebral vasodilatation with electrical stimulation of the ganglia in the rat. We had found that an orbital approach during which the eye was retracted for visualization of the ganglion precluded eliciting vasodilatation with ganglionic stimulation. In the current study approaching the ganglion through an incision over the zygomatic arch provided clear exposure of the ganglion and stimulation of the ganglion with that approach led to vasodilatation.

Parasympathetic nerves from the pterygopalatine ganglia provide nitroxidergic innervation to forebrain cerebral blood vessels. Disruption of that innervation attenuates cerebral vasodilatation seen during acute hypertension as does systemic administration of a non-selective nitric oxide synthase (NOS) inhibitor. Although such studies suggest that nitric oxide (NO) released from parasympathetic nerves participates in vasodilatation of cerebral vessels during hypertension that hypothesis has not been tested with selective local inhibition of neuronal NOS (nNOS). We tested that hypothesis through these studies performed in anesthetized rats instrumented for continuous measurement of blood pressure, heart rate and pial arterial diameter through a cranial window. We sought to determine if the nNOS inhibitor propyl-L-arginine delivered directly to the outer surface of a pial artery would 1) attenuate changes in pial arterial diameter during acute hypertension and 2) block nNOS mediated dilator effects of N-methyl-D- Aspartate (NMDA) delivered into the window but 3) not block vasodilatation elicited by acetylcholine (ACh) and mediated by endothelial NOS dilator. Without the nNOS inhibitor arterial diameter abruptly increased 70 ± 15% when mean arterial pressure (MAP) reached 183 ± 3 mmHg while with nNOS inhibition diameter increased only 13 ± 10% (p0.05). The nNOS inhibitor significantly attenuated vasodilatation induced by NMDA but not ACh delivered into the window. Thus, local nNOS inhibition attenuates breakthrough from autoregulation during hypertension as does complete interruption of the parasympathetic innervation of cerebral vessels. These findings further support the hypothesis that NO released from parasympathetic fibers contributes to cerebral vasodilatation during acute hypertension. Section: Regulatory Systems

Glutamate (GLU) receptor activation, which is important in cardiovascular reflex transmission through the nucleus tractus solitarii (NTS), leads to release of nitric oxide (NO.) from central nitroxidergic neurons. Therefore, we hypothesized that GLU and NO. are linked in cardiovascular control by NTS. We first sought to determine if NO. released into NTS led to cardiovascular changes like those produced by GLU and found that the nitrosothiol S-nitrosocysteine, but not NO. itself or other NO. donors, elicited such responses in anesthetized rats. The responses were dependent on activation of soluble guanylate cyclase but, not being affected by a scavenger of NO., likely did not depend on release of NO. into the extracellular space. Responses to ionotropic GLU agonists in NTS, like those to S-nitrosocysteine, were inhibited by inhibition of soluble guanylate cyclase. Inhibition of neuronal NO. synthase (nNOS) also inhibited responses to ionotropic GLU agonists. The apparent physiologic link between GLU and NO. mechanisms in NTS was further supported by anatomical studies that demonstrated frequent association between GLU-containing nerve terminals and neurons containing nNOS. Furthermore, GLU receptors were often found on NTS neurons that were immunoreactive for nNOS. The anatomical relationships between GLU and nNOS and GLU receptors and nNOS were more pronounced in some subnuclei of NTS than in others. While seen in subnuclei that are known to receive cardiovascular afferents, the association was even more prominent in subnuclei that receive gastrointestinal afferents. These studies support a role for nitroxidergic neurons in mediating cardiovascular and other visceral reflex responses that result from release of GLU into the NTS.

1. We tested the hypothesis that arterial baroreceptor reflexes modulate cerebrovascular tone through a pathway that connects the cardiovascular nucleus tractus solitarii with parasympathetic preganglionic neurons in the pons. 2. Anesthetized rats were used in all studies. Laser flowmetry was used to measure cerebral blood flow. We assessed cerebrovascular responses to increases in arterial blood pressure in animals with lesions of baroreceptor nerves, the nucleus tractus solitarii itself, the pontine preganglionic parasympathetic neurons, or the parasympathetic ganglionic nerves to the cerebral vessels. Similar assessments were made in animals after blockade of synthesis of nitric oxide, which is released by the parasympathetic nerves from the pterygopalatine ganglia. Finally the effects on cerebral blood flow of glutamate stimulation of pontine preganglionic parasympathetic neurons were evaluated. 3. We found that lesions at any one of the sites in the putative pathway or interruption of nitric oxide synthesis led to prolongation of autoregulation as mean arterial pressure was increased to levels as high as 200 mmHg. Conversely, stimulation of pontine parasympathetic preganglionic neurons led to cerebral vasodilatation. The second series of studies utilized classic anatomical tracing methods to determine at the light and electron microscopic level whether neurons in the cardiovascular nucleus tractus solitarii, the site of termination of baroreceptor afferents, projected to the pontine preganglionic neurons. Fibers were traced with anterograde tracer from the nucleus tractus solitarii to the pons and with retrograde tracer from the pons to the nucleus tractus solitarii. Using double labeling techniques we further studied synapses made between labeled projections from the nucleus tractus solitarii and preganglionic neurons that were themselves labeled with retrograde tracer placed into the pterygopalatine ganglion. 4. These anatomical studies showed that the nucleus tractus solitarii directly projects to pontine preganglionic neurons and makes asymmetric, seemingly excitatory, synapses with those neurons. These studies provide strong evidence that arterial baroreceptors may modulate cerebral blood flow through direct connections with pontine parasympathetic neurons. Further study is needed to clarify the role this pathway plays in integrative physiology.

Injection into the rat NTS of anti-dopamine-beta-hydroxylase-saporin (anti-DBH-SAP), which causes local loss of neuronal DBH immunoreactivity (IR), or of stabilized substance P- SAP (SSP-SAP), which causes local loss of neuronal neurokinin-1 receptor (NK1R) IR, leads to lability of arterial pressure and diminished arterial baroreflex function. We have found that anti-DBH-SAP and SSP-SAP also lead to loss of IR for glial fibrillary acidic protein (GFAP) in the NTS. To further characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we sought to compare and contrast cardiovascular effects of anti-DBH-SAP, SSP-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), 6-hydoxydopamine (6-OHDA, a neurotoxin without SAP) and PBS injected into NTS. We assessed effects of injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), GFAP, connexin 43 (Cx43), DBH, NK1R and protein gene product 9.5 (PGP 9.5)] and on arterial pressure and baroreflex function. We found that each compound containing SAP (including blank-SAP and unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP decreased TH/ DBH IR but not NK1R IR in NTS while SSP-SAP led to loss of NK1R IR as well as glutamate receptors but not DBH IR. SSP-SAP also caused PGP9.5 loss, not seen with other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 but no loss of neuronal markers. Injection of SAP alone in NTS led to significantly attenuated baroreflex responses and to significant hypertension. In contrast, 6-OHDA led to loss of TH and DBH IR and decreased Cx-43, but it increased GFAP. No baroreflex or cardiovascular effects were seen after injection of 6-OHDA, which spared glia. This study supports a critical role for NTS glia in central cardiovascular control and in maintenance of normal blood pressure.

Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex.

Lesions that remove neurons expressing neurokinin-1 (NK1) receptors from the nucleus tractus solitarii (NTS) without removing catecholaminergic neurons lead to loss of baroreflexes, labile arterial pressure, myocardial lesions, and sudden death. Because destruction of NTS catecholaminergic neurons expressing tyrosine hydroxylase (TH) may also cause lability of arterial pressure and loss of baroreflexes, we sought to test the hypothesis that cardiac lesions associated with lability are not dependent on damage to neurons with NK1 receptors but would also occur when TH neurons in NTS are targeted. To rid the NTS of TH neurons we microinjected anti-dopamine β-hydroxylase conjugated to saporin (anti-DBH-SAP, 42 ng/200 nl) into the NTS. After injection of the toxin unilaterally, immunofluorescent staining confirmed that anti-DBH-SAP decreased the number of neurons and fibers that contain TH and DBH in the injected side of the NTS while sparing neuronal elements expressing NK1 receptors. Bilateral injections in eight rats led to significant lability of arterial pressure. For example, on day 8 standard deviation of mean arterial pressure was 16.8 ± 2.5 mmHg when compared with a standard deviation of 7.83 ± 0.33 mmHg in six rats in which phosphate buffered saline (PBS) had been injected bilaterally. Two rats died suddenly at 5 and 8 days after anti-DBH-SAP injection. Seven-treated animals demonstrated microscopic myocardial necrosis as reported in animals with lesions of NTS neurons expressing NK1 receptors. Thus, cardiac and cardiovascular effects of lesions directed toward catecholamine neurons of the NTS are similar to those following damage directed toward NK1 receptor-containing neurons.

We sought to test the hypothesis that cardiovascular responses to activation of ionotropic, but not metabotropic, glutamate receptors in the nucleus tractus solitarii (NTS) depend on soluble guanylate cyclase (sGC) and that inhibition of sGC would attenuate baroreflex responses to changes in arterial pressure. In adult male Sprague-Dawley rats anesthetized with chloralose, the ionotropic receptor agonists N-methyl-d-aspartate (NMDA) and dl-α-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and the metabotropic receptor agonist trans-dl-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were microinjected into the NTS before and after microinjection of sGC inhibitors at the same site. Inhibition of sGC produced significant dose-dependent attenuation of cardiovascular responses to NMDA but did not alter responses produced by injection of AMPA or ACPD. Bilateral inhibition of sGC did not alter arterial pressure, nor did it attenuate baroreflex responses to pharmacologically induced changes in arterial pressure. This study links sGC with NMDA, but not AMPA or metabotropic, receptors in cardiovascular signal transduction through NTS.

Because inhibition of neuronal nitric oxide synthase in the nucleus tractus solitarii blocks cardiovascular responses to activation of local glutamate receptors, and because glutamate is a neurotransmitter of baroreceptor afferent nerves, we sought to test the hypothesis that neuronal nitric oxide synthase inhibition would block baroreflex transmission and cause hypertension. We determined reflex heart rate responses to intravenous phenylephrine and sodium nitroprusside in 5 anesthetized rats before and after bilateral microinjection (100 nL) of the neuronal nitric oxide synthase inhibitor AR-R 17477 (7.5 nmol) into the nucleus tractus solitarii. The inhibitor significantly increased mean arterial pressure without affecting heart rate, and it significantly reduced the gain of the baroreflex. After administration of the inhibitor, reflex responses of heart rate to changes in mean arterial pressure were always less than those responses to the same, or less, change in mean arterial pressure in the same animal without administration of the inhibitor. Microinjection of saline (100 nL) bilaterally into the nucleus tractus solitarii did not lead to hypertension or change baroreflex responses. These data support the hypothesis and suggest that neuronal nitric oxide synthase is critical to transmission of baroreflex signals through the nucleus tractus solitarii.

1. Cerebral blood flow (CBF) and cerebrovascular resistance (CVR) autoregulate to higher levels of arterial pressure (AP) in rats after sinoaortic denervation (SAD) than in intact rats. 2. Potential mechanisms for this phenomenon were studied by monitoring CBF by laser flowmetry while increasing AP in rats after: (i) SAD; (ii) SAD plus bilateral removal of the superior cervical ganglia; (iii) SAD plus interruption of all renal vessels and nerves (renal isolation); (iv) SAD plus sympathectomy and renal isolation; and (v) intravenous treatment with the nitric oxide synthase inhibitor L-nitroarginine (LNA). 3. Compared to intact control rats autoregulation persisted to higher absolute levels of AP in SAD rats with isolation of the kidneys and resistance did not fall significantly below that in rats with SAD alone. 4. Effects of SAD on autoregulation were not altered by combining renal isolation with an interruption of sympathetics. 5. LNA did not affect the baroreflex but blocked a breakthrough of autoregulation even at a maximal mean AP (MAP) of 189 +/- 2 mmHg. 6. Breakthrough occurred in animals pretreated with LNA plus L-arginine but not in animals given LNA plus D-arginine. 7. These data are consistent with a role of the baroreflex in the expression of breakthrough and suggest that breakthrough may result from release of nitric oxide or a nitric oxide donor.

1. We tested the hypothesis that arterial baroreceptor reflexes modulate cerebrovascular tone through a pathway that connects the cardiovascular nucleus tractus solitarii with parasympathetic preganglionic neurons in the pons. 2. Anesthetized rats were used in all studies. Laser flowmetry was used to measure cerebral blood flow. We assessed cerebrovascular responses to increases in arterial blood pressure in animals with lesions of baroreceptor nerves, the nucleus tractus solitarii itself, the pontine preganglionic parasympathetic neurons, or the parasympathetic ganglionic nerves to the cerebral vessels. Similar assessments were made in animals after blockade of synthesis of nitric oxide, which is released by the parasympathetic nerves from the pterygopalatine ganglia. Finally the effects on cerebral blood flow of glutamate stimulation of pontine preganglionic parasympathetic neurons were evaluated. 3. We found that lesions at any one of the sites in the putative pathway or interruption of nitric oxide synthesis led to prolongation of autoregulation as mean arterial pressure was increased to levels as high as 200 mmHg. Conversely, stimulation of pontine parasympathetic preganglionic neurons led to cerebral vasodilatation. The second series of studies utilized classic anatomical tracing methods to determine at the light and electron microscopic level whether neurons in the cardiovascular nucleus tractus solitarii, the site of termination of baroreceptor afferents, projected to the pontine preganglionic neurons. Fibers were traced with anterograde tracer from the nucleus tractus solitarii to the pons and with retrograde tracer from the pons to the nucleus tractus solitarii. Using double labeling techniques we further studied synapses made between labeled projections from the nucleus tractus solitarii and preganglionic neurons that were themselves labeled with retrograde tracer placed into the pterygopalatine ganglion. 4. These anatomical studies showed that the nucleus tractus solitarii directly projects to pontine preganglionic neurons and makes asymmetric, seemingly excitatory, synapses with those neurons. These studies provide strong evidence that arterial baroreceptors may modulate cerebral blood flow through direct connections with pontine parasympathetic neurons. Further study is needed to clarify the role this pathway plays in integrative physiology.

Autoregulation maintains cerebral blood flow near basal levels as blood pressure increases, but vasodilation, breakthrough, occurs when hypertension exceeds the autoregulatory range. Loss of breakthrough after transection of baroreceptor nerves suggests that breakthrough is neurally mediated. We hypothesize that central baroreflex interruption will likewise prevent breakthrough. In treated rats, injections of lidocaine into the nucleus tractus solitarii blocked breakthrough and the baroreflex. Therefore, central, like peripheral, baroreflex interruption extends autoregulation during hypertension.

This study tested the hypothesis that cerebral vasodilatation during marked acute hypertension is mediated in part through the influence of parasympathetic nerves from the pterygopalatine ganglia. Blood pressure was increased slowly in anesthetized rats after bilateral transection of the parasympathetic nerves. Cerebral blood flow was measured by laser flowmetry. Acutely hypertensive denervated animals developed significantly less cerebral vasodilatation than did control animals with intact nerves. Thus, parasympathetic vasodilator nerves contribute to vasodilatation seen with acute hypertension.

In anesthetized intact rats, cerebral blood flow is autoregulated until mean arterial blood pressure (MAP) exceeds 150 mmHg. At higher pressures cerebral blood flow breaks through autoregulation and rapidly increases. However, interruption of the arterial baroreceptor reflex eliminates breakthrough of autoregulation. Thus, breakthrough may reflect active rather than passive vasodilatation. We, therefore, sought to determine if breakthrough depends upon synthesis of the vasodilator nitric oxide. Thirty-eight anesthetized adult male Sprague-Dawley rats were studied. In all, MAP was raised by slow i.v. infusion of phenylephrine. In rats pretreated with the nitric oxide synthase inhibitor L-nitroarginine (LNA; 22 mg/kg i.v.) or with a combination of LNA plus D-arginine (DArg; 240 mg/kg i.v.), breakthrough did not occur even when MAP exceeded 185 mmHg (LNA) and 165 mmHg (DArg). In contrast, breakthrough occurred in rats treated with LNA plus L-arginine (LArg; 240 mg/kg i.v.) and in rats whose basal vascular tone had been increased by pretreatment with arginine vasopressin prior to infusion of phenylephrine. Removal of sympathetic innervation to cerebral vessls attenuated, but did not eliminate, effects of LNA on breakthrough. Thus, vasodilatation seen with breakthrough of autoregulation depends upon release of nitric oxide or a nitric oxide donor.