Selection of a novel cell-internalizing RNA aptamer specific for CD22 antigen in B cell acute lymphoblastic leukemia.

Despite improvements in B cell acute lymphoblastic leukemia (B-ALL) treatment, a significant number of patients experience relapse of the disease, resulting in poor prognosis and high mortality. One of the drawbacks of current B-ALL treatments is the high toxicity associated with the non-specificity of chemotherapeutic drugs. Targeted therapy is an appealing strategy to treat B-ALL to mitigate these toxic off-target effects. One such target is the B cell surface protein CD22. The restricted expression of CD22 on the B-cell lineage and its ligand-induced internalizing properties make it an attractive target in cases of B cell malignancies. To target B-ALL and the CD22 protein, we performed cell internalization SELEX (Systematic Evolution of Ligands by EXponential enrichment) followed by molecular docking to identify internalizing aptamers specific for B-ALL cells that bind the CD22 cell-surface receptor. We identified two RNA aptamers, B-ALL1 and B-ALL2, that target human malignant B cells, with B-ALL1 the first documented RNA aptamer interacting with the CD22 antigen. These B-ALL-specific aptamers represent an important first step toward developing novel targeted therapies for B cell malignancy treatments.

Delivery of Cell-Specific Aptamers to the Arterial Wall with an Occlusion Perfusion Catheter.

Current strategies to prevent restenosis following endovascular treatment include the local delivery of anti-proliferative agents to inhibit vascular smooth muscle cell (VSMC) proliferation and migration. These agents, not specific to VSMCs, are deposited on the luminal surface and therefore target endothelial cells and delay vascular healing. Cell-targeted therapies, (e.g., RNA aptamers), can potentially overcome these safety concerns by specifically binding to VSMC and inhibiting proliferation and migration. The purpose of this study was to therefore demonstrate the ability of a perfusion catheter to deliver cell-specific RNA aptamer inhibitors directly to the vessel wall. RNA aptamers specific to VSMCs were developed using an in vitro cell-based systematic evolution of ligand by exponential enrichment selection process. Two aptamers (Apt01 and Apt14) were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. Local drug delivery of the aptamers into the medial wall was accomplished using a novel perfusion catheter. We demonstrated the feasibility to deliver aptamer-based drugs directly to the medial layer of an artery using a perfusion catheter. Such cell-specific targeted therapeutic drugs provide a potentially safer and more effective treatment option for patients with vascular disease.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome.

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

Cellular Localization of Acid-Sensing Ion Channel 1 in Rat Nucleus Tractus Solitarii.

By determining its cellular localization in the nucleus tractus solitarii (NTS), we sought anatomical support for a putative physiological role for acid-sensing ion channel Type 1 (ASIC1) in chemosensitivity. Further, we sought to determine the effect of a lesion that produces gliosis in the area. In rats, we studied ASIC1 expression in control tissue with that in tissue with gliosis, which is associated with acidosis, after saporin lesions. We hypothesized that saporin would increase ASIC1 expression in areas of gliosis. Using fluorescent immunohistochemistry and confocal microscopy, we found that cells and processes containing ASIC1-immunoreactivity (IR) were present in the NTS, the dorsal motor nucleus of vagus, and the area postrema. In control tissue, ASIC1-IR predominantly colocalized with IR for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial marker, integrin αM (OX42). The subpostremal NTS was the only NTS region where neurons, identified by protein gene product 9.5 (PGP9.5), contained ASIC1-IR. ASIC1-IR increased significantly (157 ± 8.6% of control, p < 0.001) in the NTS seven days after microinjection of saporin. As we reported previously, GFAP-IR was decreased in the center of the saporin injection site, but GFAP-IR was increased in the surrounding areas where OX42-IR, indicative of activated microglia, was also increased. The over-expressed ASIC1-IR colocalized with GFAP-IR and OX42-IR in those reactive astrocytes and microglia. Our results support the hypothesis that ASIC1 would be increased in activated microglia and in reactive astrocytes after injection of saporin into the NTS.

Reduced responses to glutamate receptor agonists follow loss of astrocytes and astroglial glutamate markers in the nucleus tractus solitarii.

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.

Acid-sensing ion channel 1 and nitric oxide synthase are in adjacent layers in the wall of rat and human cerebral arteries.

Extracellular acidification activates a family of proteins known as acid-sensing ion channels (ASICs). One ASIC subtype, ASIC type 1 (ASIC1), may play an important role in synaptic plasticity, memory, fear conditioning and ischemic brain injury. ASIC1 is found primarily in neurons, but one report showed its expression in isolated mouse cerebrovascular cells. In this study, we sought to determine if ASIC1 is present in intact rat and human major cerebral arteries. A potential physiological significance of such a finding is suggested by studies showing that nitric oxide (NO), which acts as a powerful vasodilator, may modulate proton-gated currents in cultured cells expressing ASIC1s. Because both constitutive NO synthesizing enzymes, neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), are expressed in cerebral arteries we also studied the anatomical relationship between ASIC1 and nNOS or eNOS in both rat and human cerebral arteries. Western blot analysis demonstrated ASIC1 in cerebral arteries from both species. Immunofluorescent histochemistry and confocal microscopy also showed that ASIC1-immunoreactivity (IR), colocalized with the smooth muscle marker alpha-smooth muscle actin (SMA), was present in the anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA) and basilar artery (BA) of rat and human. Expression of ASIC1 in cerebral arteries is consistent with a role for ASIC1 in modulating cerebrovascular tone both in rat and human. Potential interactions between smooth muscle ASIC1 and nNOS or eNOS were supported by the presence of nNOS-IR in the neighboring adventitial layer and the presence of nNOS-IR and eNOS-IR in the adjacent endothelial layer of the cerebral arteries.

Astrocytes in the rat nucleus tractus solitarii are critical for cardiovascular reflex control.

We have shown that an antibody to dopamine-ß-hydroxylase conjugated with saporin (anti-DBH-SAP) damages catecholamine neurons in the nucleus tractus solitarii (NTS) of rat, attenuates arterial baroreflexes, and leads to lability of arterial blood pressure, damage to cardiac myocytes, and, in some animals, sudden death. However, others have shown that injection of 6-hydroxydopamine (6-OHDA), a toxin devoid of saporin, also damaged NTS catecholamine neurons but did not lead to these cardiovascular changes. We found similar cardiovascular changes after injecting a different SAP conjugate to target NTS neurons with neurokinin (NK1) receptors. Because ribosome-inactivating proteins may be toxic to glia, we hypothesized that SAP, a ribosome-inactivating protein, might target glia whose loss could account for physiological changes. We tested this hypothesis by assessing effects on select neurons and on glia in the NTS after exposure to SAP, targeted SAP conjugates, or 6-OHDA. SAP and all SAP conjugates led to loss of immunoreactivity for glial fibrillary acidic protein, a marker for astrocytes, in the NTS while 6-OHDA did not. As reported previously, anti-DBH-SAP selectively killed noradrenergic neurons in the NTS while SAP conjugated to stabilized substance P (SSP-SAP) selectively killed neurons with NK1 receptors. In contrast, SAP produced no demonstrable neuronal damage. All injections led to activation of microglia in the NTS; however, only SAP and its conjugates attenuated cardiovascular reflexes while also producing lability of arterial pressure, damage to cardiac myocytes, and in some animals, sudden death. Thus, NTS astrocytes may play a role in mediating cardiovascular reflex transmission through the NTS.

Sudden death following selective neuronal lesions in the rat nucleus tractus solitarii.

In efforts to assess baroreflex and cardiovascular responses in rats in which substance P (SP) or catecholamine transmission had been eliminated we studied animals after bilateral injections into the nucleus tractus solitarii (NTS) of SP or stabilized SP (SSP) conjugated to saporin (SP-SAP or SSP-SAP respectively) or SAP conjugated to an antibody to dopamine-ß-hydroxylase (anti-DBH-SAP). We found that SP- and SSP-SAP eliminated NTS neurons that expressed the SP neurokinin-1 receptor (NK1R) while anti-DBH-SAP eliminated NTS neurons expressing tyrosine hydroxylase (TH) and DBH. The toxins were selective. Thus SP- or SSP-SAP did not eliminate TH/DBH neurons and anti-DBH-SAP did not eliminate NK1R neurons in the NTS. Each toxin, however, led to chronic lability of arterial blood pressure, diminished baroreflex function, cardiac ventricular irritability, coagulation necrosis of cardiac myocytes and, in some animals, sudden death associated with asystole. However, when TH/DBH neurons were targeted and eliminated by injection of 6-hydroxydopamine (6-OHDA), none of the cardiovascular or cardiac changes occurred. The studies reviewed here reveal that selective lesions of the NTS lead to altered baroreflex control and to cardiac changes that may lead to sudden death. Though the findings could support a role for SP or catecholamines in baroreflex transmission neither is proven in that NK1R colocalizes with glutamate receptors. Thus neurons with both are lost when treated with SP- or SSP-SAP. In addition, loss of catecholamine neurons after treatment with 6-OHDA does not affect cardiovascular control. Thus, the effect of the toxins may depend on an action of SAP independent of the effects of the SAP conjugates on targeted neuronal types.

Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat.

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.

Sudden death and myocardial lesions after damage to catecholamine neurons of the nucleus tractus solitarii in rat.

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 ß-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.

Collateral damage and compensatory changes after injection of a toxin targeting neurons with the neurokinin-1 receptor in the nucleus tractus solitarii of rat.

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 nitric 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.

Targeting neurons of rat nucleus tractus solitarii with the gene transfer vector adeno-associated virus type 2 to up-regulate neuronal nitric oxide synthase.

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.

Nitroxidergic innervation of human cerebral arteries.

A dense network of nerves containing neuronal nitric oxide synthase is present in cerebral vessels from experimental animals. The nerves may regulate cerebrovascular tone, protect the brain from stroke, and contribute to cluster headaches in humans; but studies in humans have shown only modest nitroxidergic innervation of cerebral vessels. We tested the hypothesis that nerve fibers containing neuronal nitric oxide synthase richly innervate human cerebral arteries. We used immunohistochemical techniques at post mortem and found dense neuronal nitric oxide synthase nerve staining in human cerebral vessel walls consistent with participation of nitroxidergic fibers in human physiological and pathophysiological processes.

Glutamatergic neurons say NO in the nucleus tractus solitarii.

Both glutamate and nitric oxide (NO) may play an important role in cardiovascular reflex and respiratory signal transmission in the nucleus tractus solitarii (NTS). Pharmacological and physiological data have shown that glutamate and NO may be linked in mediating cardiovascular regulation by the NTS. Through tract tracing, multiple-label immunofluorescent staining, confocal microscopic, and electronic microscopic methods, we and other investigators have provided anatomical evidence that supports a role for glutamate and NO as well as an interaction between glutamate and NO in cardiovascular regulation in the NTS. This review article focuses on summarizing and discussing these anatomical findings. We utilized antibodies to markers of glutamatergic neurons and to neuronal NO synthase (nNOS), the enzyme that synthesizes NO in NTS neurons, to study the anatomical relationship between glutamate and NO in rats. Not only were glutamatergic markers and nNOS both found in similar subregions of the NTS and in vagal afferents, they were also frequently colocalized in the same neurons and fibers in the NTS. In addition, glutamatergic markers and nNOS were often present in fibers that were in close apposition to each other. Furthermore, N-methyl-d-aspartate (NMDA) type glutamate receptors and nNOS were often found on the same NTS neurons. Similarly, alpha-amino-3-hydroxy-5-methylisoxozole-proprionic acid (AMPA) type glutamate receptors also frequently colocalized with nNOS in NTS neurons. These findings support the suggestion that the interaction between glutamate and NO may be mediated both through NMDA and AMPA receptors. Finally, by applying tracer to the cut aortic depressor nerve (ADN) to identify nodose ganglion (NG) neurons that transmit cardiovascular signals to the NTS, we observed colocalization of vesicular glutamate transporters (VGluT) and nNOS in the ADN neurons. Thus, taken together, these neuroanatomical data support the hypothesis that glutamate and NO may interact with each other to regulate cardiovascular and likely other visceral functions through the NTS.

Cardiac damage after lesions of the nucleus tractus solitarii.

Humans with central lesions that augment sympathetic nerve activity are predisposed to cardiac arrhythmias, myocardial lesions, and sudden death. Previously, we showed that selectively killing neurons with neurokinin-1 receptors in the nucleus tractus solitarii (NTS) of rats attenuated the baroreflex and, in some animals, led to sudden unexplained death within approximately 2 wk. Interruption of arterial baroreflexes is known to increase sympathetic activity. Here we tested the hypothesis that lesions in the NTS lead to fatal cardiac arrhythmias and myocardial lesions. We studied electrocardiograms, echocardiograms, blood pressure, and heart rate in 14 adult male rats after bilateral microinjection into the NTS of stabilized substance P conjugated to the toxin saporin and compared the variables in five sham control rats and in five animals with toxin injected outside the NTS. Only injection of toxin into the NTS led to increased lability of arterial blood pressure, a sign of baroreflex interruption. Two animals treated with toxin died suddenly. All animals engaged in normal activity until, in two, rapid development of asystole and death over 6-8 min. Cardiac function when examined by echocardiography was normal, but pathologic examination of the heart revealed diffuse microscopic areas of acute coagulation necrosis in the myocardium in five animals, focal subacute necrosis in two animals, and both changes in one animal. This study supports the hypothesis that NTS lesions interrupting the baroreflex may induce cardiac arrhythmias and myocardial changes similar to those seen in humans with central lesions and may lead to sudden cardiac death.

NMDA receptors in nucleus tractus solitarii are linked to soluble guanylate cyclase.

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-alpha-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.

Evidence for a glutamatergic input to pontine preganglionic neurons of the superior salivatory nucleus in rat.

Parasympathetic preganglionic neurons of the superior salivatory nucleus (SSN), which projects to the pterygopalatine ganglion (PPG), modulate salivation, lacrimation, and cerebrovascular tone. Our previous studies suggest that excitatory projections from the nucleus tractus solitarii modulate cerebrovascular tone by actions on SSN neurons. In this study we sought to test the hypothesis that N-methyl-D-aspartate (NMDA) type glutamate receptors and vesicular glutamate transporters (VGLUT) are present in the SSN and that SSN neurons receive glutamatergic input. In six rats we injected tetramethylrhodamine dextran (TRD), a fluorescent tracer, unilaterally into the PPG to label SSN neurons. Four days later, rats were perfused and brain stem sections containing the SSN were processed for fluorescent immunohistochemistry for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) and vesicular glutamate transporters (VGLUT1 and VGLUT2). Confocal laser scanning microscopy showed that 88+/-3% of TRD-labeled SSN neurons contained NMDAR1-immunoreactivity (IR). The surrounding neuropil contained numerous fibers labeled for VGLUT2-IR, but not VGLUT1-IR. Double fluorescent immunohistochemistry for NMDAR1 and VGLUT2 revealed that fibers containing VGLUT2-IR were often in close proximity to cell bodies or proximal dendrites of TRD-labeled SSN neurons that were positive for NMDAR1-IR. These studies support our hypothesis that NMDA receptors and VGLUT are present in the SSN. They further provide support for the suggestion that there are glutamatergic inputs to SSN neurons and would be consistent with an excitatory input that could regulate cerebrovascular tone.

Ablation of NK1 receptors in rat nucleus tractus solitarii blocks baroreflexes.

The neuropeptide substance P (SP) is found in vagal afferent nerves within the nucleus tractus solitarii, where it is released on stimulation of arterial baroreflexes. The neurokinin-1 receptors at which SP may act have been identified in the nucleus tractus solitarii, but there remains uncertainty if the neurons at which SP acts are critical to baroreflex transmission. By using SP conjugated with the toxin saporin, which kills the neurons at which SP may act, we sought to test the hypothesis that neurons expressing the neurokinin-1 receptor are critical to baroreflex transmission in the nucleus tractus solitarii. One and 2 weeks after injection of the toxin into the rat nucleus tractus solitarii, immunoreactivity for the neurokinin-1 receptor was lost. When the toxin had been injected bilaterally, the baroreflex gain was significantly reduced. Therefore, neurons that express SP receptors play a critical role in mediating baroreflexes through the nucleus tractus solitarii of rat.

Coexistence of NMDA and AMPA receptor subunits with nNOS in the nucleus tractus solitarii of rat.

We previously showed that most neuronal nitric oxide synthase (nNOS)-containing neurons in the nucleus tractus solitarii (NTS) contain NMDAR1, the fundamental subunit for functional N-methyl-D-aspartate (NMDA) receptors. Likewise, we found that almost all nNOS-containing neurons in the NTS contain GluR1, the calcium permeable AMPA receptor subunit. These data suggest that AMPA and NMDA receptors may colocalize in NTS neurons that contain nNOS. However, other investigators have suggested that non-NMDA receptors are located primarily on second-order neurons and NMDA receptors are located predominantly on higher-order neurons in NTS. We now seek to test the hypothesis that NMDA receptors, AMPA receptors and nNOS are colocalized in NTS cells. We performed triple fluorescent immunohistochemical staining of nNOS, NMDAR1 and GluR1, and performed confocal laser scanning microscopic analysis of the NTS. The distributions of nNOS immunoreactivity (IR), NMDAR1-IR and GluR1-IR in the NTS were similar to those we reported earlier. Superimposed images revealed that almost all NMDAR1-IR cells contained GluR1-IR and almost all GluR1-IR cells contained NMDAR1-IR. Some double-labeled cells were additionally labeled for nNOS-IR. All nNOS-IR neurons contained both GluR1-IR and NMDAR1-IR. These studies support our hypothesis that NMDA and AMPA receptors are colocalized in NTS neurons and are consistent with a role of both types of ionotropic receptors in transmission of afferent signals in NTS. In addition, these data provide support for an anatomical link between ionotropic glutamate receptors and nitric oxide in the NTS.