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.

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.

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.

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.

Neuronal nitric oxide mediates cerebral vasodilatation during acute hypertension.

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 mm Hg while with nNOS inhibition diameter increased only 13+/-10% (p<0.05) even when MAP reached 191+/-4 mm Hg (p>0.05). 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.

Parasympathetic stimulation elicits cerebral vasodilatation in rat.

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.

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.

Direct projections from the cardiovascular nucleus tractus solitarii to pontine preganglionic parasympathetic neurons: a link to cerebrovascular regulation.

Peripheral or central interruption of the baroreflex or the parasympathetic innervation of cerebral vessels leads to similar changes in regulation of cerebral blood flow. Therefore, we sought to test the hypothesis that the cardiovascular nucleus tractus solitarii, the site of termination of arterial baroreceptor nerves, projects to pontine preganglionic neurons whose stimulation elicits cerebral vasodilatation. The current study utilized both light and electron microscopic techniques to analyze anterograde tracing from the cardiovascular nucleus tractus solitarii to preganglionic parasympathetic neurons in the pons. We further used retrograde tracing from that same pontine region to the cardiovascular nucleus tractus solitarii and evaluated the confluence of tracing from the cardiovascular nucleus tractus solitarii to pontine preganglionic neurons labeled retrogradely from the pterygopalatine ganglia. The cardiovascular nucleus tractus solitarii projected to pontine preganglionic parasympathetic neurons, but more rostral and caudal regions of nucleus tractus solitarii did not. In contrast, all three regions of nucleus tractus solitarii projected to the nucleus ambiguus and dorsal motor nucleus of the vagus. Although not projecting to pontine preganglionic parasympathetic neurons, regions lateral, rostral, and caudal to cardiovascular nucleus tractus solitarii sent projections through the pons medial to the preganglionics. The study establishes the presence of a direct monosynaptic pathway from neurons in the cardiovascular nucleus tractus solitarii to pontine preganglionic parasympathetic neurons that project to the pterygopalatine ganglia, the source of nitroxidergic vasodilatory innervation of cerebral blood vessels. It provides evidence that activation of those preganglionic neurons can cause cerebral vasodilatation and increased cerebral blood flow. Finally, it demonstrates differential innervation of medullary and pontine preganglionic parasympathetic neurons by different regions of the nucleus tractus solitarii.

Comparative effects of clonidine and dihydroergotamine on venomotor tone and orthostatic tolerance in patients with severe hypoadrenergic orthostatic hypotension.

PURPOSE: Clonidine, an alpha(2)-adrenergic agonist, raises blood pressure in patients with autonomic failure, in whom failure of reflex neurogenic venoconstriction leads to severe orthostatic hypotension. Because animal studies suggest that postjunctional alpha(2)-adrenoreceptors are located mainly on venous capacitance rather than arterial resistance vessels, we tested the hypothesis that venoconstriction is the main mechanism by which clonidine raises blood pressure in patients with autonomic failure. SUBJECTS AND METHODS: We measured forearm venous and arterial tone using plethysmography in 4 patients with autonomic failure before and after acute administration of clonidine (0.4 mg orally) or dihydroergotamine (0.15 mg intravenously), a known venoconstrictor agent. We also recorded supine intraarterial pressure at rest and during graded orthostatic stress with lower body negative pressure. RESULTS: Clonidine and dihydroergotamine caused similar increases in supine (mean +/- SD) arterial pressure (+23 +/- 11 mm Hg vs. and +27 +/- 5 mm Hg) and forearm vascular resistance (+36% +/- 13% vs. +28% +/- 9%). However, the drugs had different effects on forearm venous tone, which increased by 38% +/- 9% with dihydroergotamine (P = 0.01 vs. control) but was unaffected by clonidine (change = 0% +/- 14%). A single dose of clonidine was less effective than a single dose of dihydroergotamine in maintaining arterial pressure during graded orthostatic stress. CONCLUSION: In contrast with what has been hypothesized, clonidine appears to function mainly as an arterial constrictor in patients with hypoadrenergic orthostatic hypotension. Further studies are needed to determine if venoconstrictor agents are of greater therapeutic benefit in this condition than are pure arterial vasoconstrictors.

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.

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.

Inhibiting the nucleus tractus solitarii extends cerebrovascular autoregulation during hypertension.

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.

Effect of substance P injection into the nucleus tractus solitarius of rats on cricothyroid and thyroarytenoid motor activity and cardiovascular and respiratory systems.

Identification of central neurotransmitters that mediate laryngeal adductor and/or tensor activity may prove useful in managing pathological laryngeal adduction as occurs in laryngospasm or apparent life-threatening events. The putative transmitter substance P (SP) is found in the nucleus tractus solitarius (NTS), in which laryngeal afferents terminate. Therefore, we studied the laryngeal, cardiovascular, and respiratory effects of SP injected into the NTS of rats. We completed bilateral stereotactic injections of 20 nL of SP (15 micromol) or control solution into the region of the NTS, the dorsal motor nucleus (DMN), or the nucleus gracilis (GR) in 30 anesthetized rats. Changes in diaphragm, cricothyroid (CT), and thyroarytenoid (TA) electromyography (EMG), as well as blood pressure (BP), were compared. The injection sites were verified histologically. Injection of SP into the NTS altered CT and/or TA EMG activity in all animals. The change ranged from complete inhibition, to a phasic increase, to a tonic increase. No change in laryngeal adductor EMG activity was seen in 8 of 9 animals after SP injections into the DMN (4/5) or GR (4/4), but 1 animal demonstrated brief inhibition of CT and TA EMG activity after SP injection into the DMN. Injection of SP into the NTS induced central apnea and a significant decrease in BP in all animals. The duration of apnea tended to be longer after NTS injections than after DMN or GR injections (p < .10 and p < .05, respectively). We conclude that stereotactic injections of putative neurotransmitters in rats may be accomplished to identify effects on laryngeal motor activity. Direct application of SP into the NTS consistently elicits a change in CT and/or TA EMG activity, ranging from inhibition to excitation. This model may prove useful in evaluating pharmacological targets of central reflex activity to manage life-threatening laryngeal reflex activity.

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.

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.

Views of translational research from a somewhat translational scientist.

This review arose from a talk entitled "Identifying Targets" and given by the author at EB2011 at the invitation of the American Federation for Medical Research (AFMR). The presentation was part of the American Federation for Medical Research workshop entitled "Keys for Translation: Science and Strategy" and focused on identifying clinically relevant targets as a result of observations made during basic scientific studies. The review emphasizes that targets do not have to be the aim that drives basic discovery, but communication between the basic scientist and clinical investigators may aid recognition of such targets and their translation to clinical applications. Using one line of investigator-initiated research from his own laboratory as an example, the author emphasizes that basic discovery must be hypothesis driven and allowed to follow its logical sequence. Finding treatments, while always an aim of biomedical research, may arise as a result of basic studies that were not originally aimed at a target of translational research.

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.

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.