NTS Injection Research Articles

BDNF downregulates β-adrenergic receptor-mediated hypotensive mechanisms in the paraventricular nucleus of the hypothalamus.

Brain-derived neurotrophic factor (BDNF) is upregulated in the paraventricular nucleus of the hypothalamus (PVN) in response to hypertensive stimuli such as stress and hyperosmolality, and BDNF acting in the PVN plays a key role in elevating sympathetic activity and blood pressure. However, downstream mechanisms mediating these effects remain unclear. We tested the hypothesis that BDNF increases blood pressure, in part by diminishing inhibitory hypotensive input from nucleus of the solitary tract (NTS) catecholaminergic neurons projecting to the PVN. Male Sprague-Dawley rats received bilateral PVN injections of viral vectors expressing either green fluorescent protein (GFP) or BDNF and bilateral NTS injections of vehicle or anti-dopamine-β-hydroxylase-conjugated saporin (DSAP), a neurotoxin that selectively lesions noradrenergic and adrenergic neurons. BDNF overexpression in the PVN without NTS lesioning significantly increased mean arterial pressure (MAP) in awake animals by 18.7 ± 1.8 mmHg. DSAP treatment also increased MAP in the GFP group, by 9.8 ± 3.2 mmHg, but failed to affect MAP in the BDNF group, indicating a BDNF-induced loss of NTS catecholaminergic hypotensive effects. In addition, in α-chloralose-urethane-anesthetized rats, hypotensive responses to PVN injections of the β-adrenergic agonist isoprenaline were significantly attenuated by BDNF overexpression, whereas PVN injections of phenylephrine had no effect on blood pressure. BDNF treatment was also found to significantly reduce β1-adrenergic receptor mRNA expression in the PVN, whereas expression of other adrenergic receptors was unaffected. In summary, increased BDNF expression in the PVN elevates blood pressure, in part by downregulating β-receptor signaling and diminishing hypotensive catecholaminergic input from the NTS to the PVN.NEW & NOTEWORTHY We have shown that BDNF, a key hypothalamic regulator of blood pressure, disrupts catecholaminergic signaling between the NTS and the PVN by reducing the responsiveness of PVN neurons to inhibitory hypotensive β-adrenergic input from the NTS. This may be occurring partly via BDNF-mediated downregulation of β1-adrenergic receptor expression in the PVN and results in an increase in blood pressure.

Inhibition of glial glutamate transporter GLT1 in the nucleus of the solitary tract attenuates baroreflex control of sympathetic nerve activity and heart rate.

The astrocytic glutamate transporter (GLT1) plays an important role in the maintenance of extracellular glutamate concentration below neurotoxic levels in brain. However, the functional role of GLT1 within the nucleus of the solitary tract (NTS) in the regulation of cardiovascular function remains unclear. We examined the effect of inhibiting GLT1 in the subpostremal NTS on mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA) and heart rate (HR) in anesthetized, artificially ventilated rats. It was found that dihydrokainate (DHK; inhibitor of GLT1, 5 mmol/L, 100 nL) injections into the NTS (n = 6) decreased MAP (50 ± 10 mmHg, mean ± SD), RSNA (89 ± 14%) and HR (37 ± 6 bpm). Pretreatment with kynurenate (KYN; glutamate receptor antagonist, 5 mmol/L, 30 μL) topically applied to the dorsal surface of the brainstem (n = 4) attenuated the responses to NTS injections of DHK (P < 0.01). The effect of DHK on arterial baroreflex function was examined using i.v. infusions of phenylephrine and nitroprusside. DHK reduced baroreflex response range (maximum−minimum) of RSNA by 91 ± 2% and HR by 83 ± 5% (n = 6, P < 0.001). These results indicate that inhibition of GLT1 within the NTS decreases MAP, RSNA, and HR by the activation of ionotropic glutamate receptors. As a result, baroreflex control of RSNA and HR was dramatically attenuated. The astrocytic glutamate transporter in the NTS plays an important role in the maintenance and regulation of cardiovascular function.

BDNF Downregulates Adrenergic b‐Receptor‐Mediated Hypotensive Mechanisms in the Paraventricular Nucleus of the Hypothalamus (PVN)

Brain derived neurotrophic factor (BDNF) is an important modulator of neuronal function, capable of mediating long‐term changes in neuronal structure and signaling in the central nervous system (CNS). Increased expression of BDNF in the PVN has been associated with elevated blood pressure (BP) and sympathetic nervous system activity. However, the mechanism mediating this effect of BDNF is unclear. BDNF is a modulator of catecholaminergic (CA‐ergic) neuronal function in the CNS, and could potentially influence CA‐ergic input to the PVN. The majority of CA‐ergic projections to the PVN come from the nucleus of the solitary tract (NTS), and these projections have been shown to exert a hypotensive effect. Here, we tested the hypothesis that increased BDNF expression in the PVN elevates BP in part by diminishing the inhibitory input from NTS CA‐ergic neurons projecting to the PVN by downregulating β‐receptors in the PVN. Sprague‐Dawley (SD) rats received bilateral PVN injections of AAV2 viral vectors expressing either green fluorescent protein (GFP) or BDNF and bilateral NTS injections of phosphate‐buffered saline (PBS) or anti‐dopamine‐β‐hydroxylase‐conjugated saporin (DSAP), a neurotoxin selective to noradrenaline‐ and adrenaline‐synthesizing neurons. BDNF overexpression in the PVN without lesioning NTS CAergic neurons significantly increased mean arterial pressure (MAP) (BDNF+PBS: 115±3 mmHg, p&lt;0.01 vs GFP+PBS: 96±2 mmHg). DSAP treatment increased MAP in the GFP group by ~13 mmHg, whereas DSAP treatment in the BDNF group did not significantly alter MAP. This suggests that BDNF overexpression in the PVN may interfere with CA‐ergic communication between the NTS and PVN. Since previous reports suggested that adrenergic β‐receptors exert an inhibitory effect on PVN neurons and lower BP, we tested whether BDNF overexpression in the PVN diminishes hypotensive effects of β‐receptor activation in the PVN. SD rats received bilateral PVN injections of AAV2 viral vectors expressing either GFP or BDNF. Three weeks later, BP responses to an injection of Isoprenaline (125 μM or 250 μM), a non‐selective β‐adrenergic agonist, into the PVN were recorded under alpha chloralosed‐urethane anesthesia. Our results showed that BDNF treatment significantly attenuated MAP responses to Isoprenaline compared to the GFP group in a dose dependent manner. In the GFP group, peak decrease in MAP in response to 125 μM and 250 μM Iso was −21±4 mmHg and −29±4 mmHg, compared with −4±1 mmHg (p&lt;0.01) and −8±1 mmHg (p&lt;0.001) in the BDNF group. To test if the reduced effect of Isoprenaline is due to a BDNF‐induced change in adrenergic receptor expression in the PVN, we assessed adrenergic receptor expression using quantitative RT‐PCR in PVN brain punches from SD rats previously injected with an AAV2 viral vectors expressing either GFP or BDNF. BDNF treatment significantly reduced the expression of β1 adrenergic receptor mRNA expression, whereas α1a, α1b, α2a, and β2 adrenergic receptors were unaffected by BDNF treatment. In summary, our findings indicate that increased BDNF expression in the PVN may disrupt CA‐ergic signaling between the NTS and PVN by downregulating β‐receptors in the PVN.Support or Funding InformationSupported by R01 HL133211‐01A1, AHA 11SDG7560022, and UVM start‐up funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcription factor ΔFosB acts within the nucleus of the solitary tract to increase mean arterial pressure during exposures to intermittent hypoxia.

ΔFosB is a member of the activator protein-1 family of transcription factors. ΔFosB has low constitutive expression in the central nervous system and is induced after exposure of rodents to intermittent hypoxia (IH), a model of the arterial hypoxemia that accompanies sleep apnea. We hypothesized ΔFosB in the nucleus of the solitary tract (NTS) contributes to increased mean arterial pressure (MAP) during IH. The NTS of 11 male Sprague-Dawley rats was injected (3 sites, 100 nl/site) with a dominant negative construct against ΔFosB (ΔJunD) in an adeno-associated viral vector (AAV)-green fluorescent protein (GFP) reporter. The NTS of 10 rats was injected with AAV-GFP as sham controls. Two weeks after NTS injections, rats were exposed to IH for 8 h/day for 7 days, and MAP was recorded using telemetry. In the sham group, 7 days of IH increased MAP from 99.8 ± 1.1 to 107.3 ± 0.5 mmHg in the day and from 104.4 ± 1.1 to 109.8 ± 0.6 mmHg in the night. In the group that received ΔJunD, IH increased MAP during the day from 95.9 ± 1.7 to 101.3 ± 0.4 mmHg and from 100.9 ± 1.7 to 102.8 ± 0.5 mmHg during the night (both IH-induced changes in MAP were significantly lower than sham, P < 0.05). After injection of the dominant negative construct in the NTS, IH-induced ΔFosB immunoreactivity was decreased in the paraventricular nucleus ( P < 0.05); however, no change was observed in the rostral ventrolateral medulla. These data indicate that ΔFosB within the NTS contributes to the increase in MAP induced by IH exposure. NEW & NOTEWORTHY The results of this study provides new insights into the molecular mechanisms that mediate neuronal adaptations during exposures to intermittent hypoxia, a model of the hypoxemias that occur during sleep apnea. These adaptations are noteworthy as they contribute to the persistent increase in blood pressure induced by exposures to intermittent hypoxia.

Abstract P189: Angiotensin II - Endocannabinoid Interactions in the Nucleus of the Solitary Tract are Important for Regulation of Baroreflex Control of Heart Rate

Hypertension resulting from elevated brain angiotensin (Ang) II is associated with impaired functioning of neural reflexes regulating sympathetic and parasympathetic outflow. Restoration of normal baroreflex sensitivity (BRS) for control of heart rate (HR) is achieved in a rat model of Ang II - dependent hypertension [ (mRen2)27 transgenic rats ] by local injection of the cannabinoid CB 1 receptor antagonist rimonabant (SR141716A), a CB 1 receptor antagonist, into the solitary tract nucleus (NTS) of anesthetized rats or by chronic oral rimonabant treatment, which has central and peripheral sites of action. Together with elevated brain dorsal medullary tissue concentrations of 2-arachidonylglycerol present in the (mRen2)27 rats, these findings are consistent with an activated endocannabinoid system contributing to the impaired BRS in these animals. To further explore acute interactions between Ang II - mediated suppression of BRS and the endocannabinoids, Ang II was injected into NTS of anesthetized Sprague-Dawley rats 10 minutes following NTS injection of rimonabant or aCSF (120 nL, bilaterally). In the presence of aCSF, Ang II reduced BRS by ~50% (In msec/mm Hg: 1.14 ± 0.14 before versus 0.53 ± 0.16; n = 7, p &lt; 0.008); this effect was abolished in the presence of rimonabant (In msec/mm Hg: 0.92 ± 0.16 before versus 0.86 ± 0.21; n = 4, p = 0.8). There was no difference in mean arterial pressure or heart rate before or after Ang II treatment in either aCSF or rimonabant groups. Thus, the data support the interpretation that Ang II - mediated attenuation of BRS for control of HR involves release of endocannabinoids. Others report that the pressor actions following acute local injections of Ang II into the hypothalamic paraventricular nucleus are prevented by blockade of CB 1 receptors. We conclude that functional interactions between these two systems occur at multiple brain sites relevant to blood pressure control mechanisms and together the findings support a role for elevated brain endocannabinoids as contributors to the altered reflexes characteristic of Ang II - dependent hypertension. Support: HL-51952, DA-024863 and DA-03690, the Hypertension &amp; Vascular Research Center, Farley[[Unable to Display Character: &amp;#8208;]]Hudson Foundation

NMDA-type glutamate receptors participate in reduction of food intake following hindbrain melanocortin receptor activation.

Hindbrain injection of a melanocortin-3/4 receptor agonist, MTII, reduces food intake primarily by reducing meal size. Our previously reported results indicate that N-methyl-D-aspartate-type glutamate receptors (NMDAR) in the nucleus of the solitary tract (NTS) play an important role in the control of meal size and food intake. Therefore, we hypothesized that activation of NTS NMDARs contribute to reduction of food intake in response to fourth ventricle or NTS injection of MTII. We found that coinjection of a competitive NMDAR antagonist (d-CPP-ene) with MTII into the fourth ventricle or directly into the NTS of adult male rats attenuated MTII-induced reduction of food intake. Hindbrain NMDAR antagonism also attenuated MTII-induced ERK1/2 phosphorylation in NTS neurons and prevented synapsin I phosphorylation in central vagal afferent endings, both of which are cellular mechanisms previously shown to participate in hindbrain melanocortinergic reduction of food intake. Together, our results indicate that NMDAR activation significantly contributes to reduction of food intake following hindbrain melanocortin receptor activation, and it participates in melanocortinergic signaling in NTS neural circuits that mediate reduction of food intake.

Time course of changes in glutamatergic transmission within NTS during CIH exposure and the role of ΔFosB (710.6)

ΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant‐negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p&lt;.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV‐GFP‐ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post‐synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement.Grant Funding Source: HL088052

Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure

Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which l-glutamate (0.25M; 10nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207±78), 32.3±2.3% co-expressed L-Enk immunoreactivity and of these, 2.6±1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo-medullary pathway may represent the anatomical substrate by which hcrt-1/L-Enk neurons function in the coordination of autonomic-cardiovascular responses during different behavioral states.

Nucleus of the Solitary Tract (Pro)Renin Receptor-Mediated Antihypertensive Effect Involves Nuclear Factor-κB-Cytokine Signaling in the Spontaneously Hypertensive Rat

The importance of the (pro)renin receptor (PRR) in the function of the central nervous system is increasingly evident because PRR seems to play a role in neuronal control of cardiovascular function. PRR expression is elevated in the nucleus of the solitary tract (NTS) of spontaneously hypertensive rats (SHR). In this study, we tested the hypothesis that altered activity of PRR in the NTS is linked to hypertension. Eight weeks of chronic knockdown of the NTS PRR, using recombinant adeno-associated virus type 2 (AAV2)-PRR-small hairpain RNA (shRNA)-mediated gene transduction, caused a significant increase in mean arterial pressure (MAP) in the SHR (shRNA, 173±5; Control, 151±6 mm Hg) but not in Wistar Kyoto rats (shRNA, 108±7; Control, 106±6 mm Hg). The MAP elevation in the SHR was associated with decreased inflammatory markers tumor necrosis factor-α, interleukin-6, C-C motif ligand 5, and their transcription factor, nuclear factor-κB. Consistent with the pressor effects of the PRR knockdown, acute bilateral NTS injection of human renin (2 pmol/side) decreased MAP and heart rate (HR) in SHR (ΔMAP, -38±4 mm Hg; Δheart rate, -40±10 bpm), with negligible responses in Wistar Kyoto rats (ΔMAP, -4±3 mm Hg; Δheart rate, -12±7 bpm). These effects in SHR were attenuated (80%) by prorenin handle region peptide but were not affected by angiotensin II type 1 or angiotensin II type 2 receptor blockers. Finally, PRR activation in SHR neuronal cultures by prorenin activated nuclear factor-κB and increased mRNA levels of interleukin-1β (250-fold), tumor necrosis factor-α (32-fold), interleukin-6 (35-fold), C-C motif ligand 5 (12-fold), and interleukin-10 (7-fold) in a nuclear factor-κB-dependent but angiotensin II type 1 receptor-independent manner. Therefore, NTS PRR mediates antihypertensive effects via an angiotensin II-independent mechanism in SHR, which involves stimulation of the nuclear factor-κB-cytokine signaling pathway.

Abstract 418: Mineralocorticoid Receptor in Nucleus of the Solitary Tract Contributes to Aldosterone Stimulation of Salt Intake

A group of neurons in the nucleus of the solitary tract (NTS) contains the mineralocorticoid receptor (MR) which makes these neurons candidates for stimulating salt intake in response to aldosterone. The purpose of this study was to determine if neurons within the NTS that possess the MR play a role in aldosterone stimulation of salt intake. Five adult WKY rats received microinjections into the NTS of a small, hairpin RNA (shRNA) for the MR (Genedetect, Inc.) and 5 rats received NTS injections of a scrambled RNA (scRNA). In each rat injections of viral constructs were made at 3 points, all 0.5mm below the surface of the brain: calamus; bilaterally at 0.5mm rostral to calamus and 0.5mm lateral to the midline. One week after the viral construct injections, aldosterone-filled osmotic mini-pumps were implanted subcutaneously and connected to tubing within the 4 th ventricle to infuse aldosterone at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl respectively. Fluid level within each bottle was measured at the same time every day and salt intake expressed as 100 X the ratio of 0.3M NaCl intake to total fluid intake. Previous studies indicate that the viral constructs require 2 weeks to have a discernible effect. One week after injection of the viral constructs and prior to infusion of implantation of the aldosterone mini-pump, salt intake in shRNA injected rats was 6% ± 3% and in scRNA injected rats it was 4% ± 2%. One week after beginning aldosterone infusion (two weeks after injection of viral constructs), salt intake in shRNA injected rats was 16% ± 5% and in scRNA injected rats it was 22% ± 7% (p=0.01). Three weeks after beginning aldosterone infusion, salt intake in shRNA injected rats was 5% ± 2% and in scRNA injected rats it was 14% ± 9% (p=0.01). Post-mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (shRNA 2 ± 2 cells/section; scRNA 12 ± 2 cells/section; p=.008). These results indicate hindbrain infusions of aldosterone stimulate salt intake and that at least part of the hindbrain aldosterone stimulation of salt intake are mediated by hindbrain NTS neurons that possess the MR.

Angiotensin type 2 receptors (AT2R) over expression in the nucleus of the solitary tract (NTS) attenuate renovascular hypertension

We have demonstrated that AT2R overexpression in the NTS decreases mean arterial pressure (MAP) in normotensive rats. The aim of the present study was to investigate the effects of AT2R overexpression in the NTS in two kidney 1 clip (2K1C) hypertensive rats. Holtzman male rats (150–180 g, n = 5–6/group) were implanted with telemetry transducers into the abdominal aorta followed by a silver clip implant in the left renal artery. Three weeks after the 2K1C surgery the NTS was microinjected (200 nl/site, 5 sites) with either adeno‐associated virus vector AAV2‐CBA‐AT2R (1×108 gc) or AAV2‐CBA‐eGFP (1×109 gc) for the over expression of AT2R or GFP, respectively. At that time period, rats were hypertensive (134±3 mmHg light period and 133±4 mmHg dark period) compared to baseline levels (light period: 101±3 mmHg; dark period: 110±5 mmHg). Three to 4 weeks after the NTS injections (6–7 weeks after 2K1C), at which time AT2R or GFP expression has reached maximal (and stable) levels, MAP reached values of 171±10 mmHg light period and 190±8 mmHg dark period in GFP rats. In the AT2R transduced 2K1C rats, the increase in MAP was significantly lower compared to GFP animals (151±6 mmHg light period; 153±7 mmHg dark period: p&lt;0.05 vs. GFP group). These results suggest that overexpression of AT2R in the NTS impairs the development of renovascular hypertension.Supported by: PNPD/CAPES, CNPq and NIH Grant HL 076803

NTS (pro)renin receptor (PRR)‐mediated antihypertensive effect involves NF‐KappaB‐cytokine signaling in the spontaneously hypertensive rats (SHR)

Increased PRR expression in the NTS of SHR, coupled with the evidence on role of the brain inflammation in cardiovascular functions, has led us to hypothesize that the NTS PRR may play a part in blood pressure (BP) regulation by modulation of the brain inflammatory pathways. Acute NTS injections of renin (0.02–2 pmol) resulted in a significantly greater dose‐dependent decrease in systolic BP (ΔSBP; −12±5 to −32±5 mmHg) and heart rate (ΔHR; −13±7 to −40±10 bpm) in the SHR compared to the WKY rats (ΔSBP: −3±1 to−8±4 mmHg; ΔHR: −2±4 to −12±7 bpm). These effects were not blocked by the AT1R or AT2R blocker. Consistent with this, chronic PRR knockdown with AAV2‐PRR‐shRNA in the SHR NTS caused an increase in mean arterial pressure (shRNA: 173±5; Control: 151±6 mmHg), and decreases in baroreflex bradycardia (shRNA: 0.26±0.04; Control: 0.44±0.04) and parasympathetic tone (shRNA: 31±4; Control: 49±6). This was accompanied by a decrease in the inflammatory markers TNFa, IL6 and Ccl5, and their transcription factors, NF‐κB and AP‐1. Finally, PRR activation in the SHR brain neuronal cultures activated NF‐κB, and increased the mRNA of IL1β (250 fold), TNFα (32 fold), IL6 (35 fold) and Ccl5 (12 fold) in an NF‐κB‐ and AP1‐dependent, but AT1R/AT2R‐independent manner. Therefore, the NTS PRR mediates Ang II‐independent antihypertensive effects by stimulation of NF‐κB/AP‐1/cytokine signaling (AHA 11SDG7420029)

Reduction of food intake by cholecystokinin requires activation of hindbrain NMDA-type glutamate receptors

Intraperitoneal injection of CCK reduces food intake and triggers a behavioral pattern similar to natural satiation. Reduction of food intake by CCK is mediated by vagal afferents that innervate the stomach and small intestine. These afferents synapse in the hindbrain nucleus of the solitary tract (NTS) where gastrointestinal satiation signals are processed. Previously, we demonstrated that intraperitoneal (IP) administration of either competitive or noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists attenuates reduction of food intake by CCK. However, because vagal afferents themselves express NMDA receptors at both central and peripheral endings, our results did not speak to the question of whether NMDA receptors in the brain play an essential role in reduction of feeding by CCK. We hypothesized that activation of NMDA receptors in the NTS is necessary for reduction of food intake by CCK. To test this hypothesis, we measured food intake following IP CCK, subsequent to NMDA receptor antagonist injections into the fourth ventricle, directly into the NTS or subcutaneously. We found that either fourth-ventricle or NTS injection of the noncompetitive NMDA receptor antagonist MK-801 was sufficient to inhibit CCK-induced reduction of feeding, while the same antagonist doses injected subcutaneously did not. Similarly fourth ventricle injection of d-3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphoric acid (d-CPPene), a competitive NMDA receptor antagonist, also blocked reduction of food intake following IP CCK. Finally, d-CPPene injected into the fourth ventricle attenuated CCK-induced expression of nuclear c-Fos immunoreactivity in the dorsal vagal complex. We conclude that activation of NMDA receptors in the hindbrain is necessary for the reduction of food intake by CCK. Hindbrain NMDA receptors could comprise a critical avenue for control and modulation of satiation signals to influence food intake and energy balance.

Modulation of Baroreflex Sensitivity by Endogenous Angiotensins in Lamb Solitary Tract Nucleus

We and others find that within the nucleus of the solitary tract (NTS) of the rat endogenous Ang II attenuates while Ang‐(1–7) facilitates the baroreflex sensitivity (BRS) in the control of heart rate. It is not known whether this same relationship exists in the sheep NTS. Therefore, we performed microinjection studies in 6 wk old sheep (n = 5) under chloralose‐urethane anesthesia (35/750 mg/kg) which maintains both increases and decreases in BRS for heart rate control. To assess the effects of blockade of either the AT1 or Ang‐(1–7) (AT7) receptors, we tested BRS before and after bilateral NTS injection of the AT1 antagonist candesartan (CV, 200 pmol/200 nl at 4 injection sites) and retested the reflex again after the AT7 antagonist D‐Ala7‐Ang‐(1–7) (D‐Ala, 10 pmol/200 nl at 4 injection sites). BRS was tested using various doses of phenylephrine to determine the slope of the relationship between changes in mean arterial pressure (MAP) and pulse interval as ms/mm Hg. Resting BRS was 0.3 ± 0.2 ms/mm Hg and MAP was 81 ± 7 mm Hg. NTS injection of CV increased BRS to a new baseline of 1.0 ± 0.2 ms/mm Hg, (p&lt;0.05). From this elevated level D‐Ala significantly reduced BRS back to baseline. There was no effect of either agent on MAP. These data suggest that baseline baroreceptor reflex control of heart rate in lambs is regulated by Ang II and Ang‐(1–7) in the NTS in an opposite manner. Support: HD‐047584, HD‐017644, HL‐51952.

Angiotensin-(1-7) and Baroreflex Function in Nucleus Tractus Solitarii of (mRen2)27 Transgenic Rats

Endogenous angiotensin (Ang)-(1-7) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)27 renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(1-7) in brain medullary, and associated with hypertension and impaired BRS. We therefore tested the contribution of endogenous Ang-(1-7) to BRS for control of HR and responses to cardiac vagal chemosensitive afferent fiber activation (CVA) with phenylbiguanide (PBG) in anesthetized SD and (mRen2) 27 rats before and after bilateral nucleus of the solitary tract (nTS) injection of the Ang-(1-7) receptor antagonist (D-Ala7)-Ang-(1-7). (mRen2) 27 rats exhibited a approximately 50% impairment in BRS as compared with SD (P < 0.05). (D-Ala7)-Ang-(1-7) attenuated BRS by approximately 50% in SD rats, but was without effect in (mRen2) 27 rats. (D-Ala7)-Ang-(1-7) did not alter the responses to CVA by PBG (iv bolus) in either strain. There were no differences in the depressor effects of Ang-(1-7) injected into the nTS, nor were levels of mRNA different for angiotensin-converting enzyme, angiotensin-converting enzyme 2, neprilysin, or the mas receptor in medullary tissue from SD versus (mRen2)27 rats. Endogenous Ang-(1-7) does not provide tonic input in the nTS to modulate BRS for control of HR in (mRen2)27 rats, which may contribute to impairment of BRS in these animals.

Inhibition of the PI3 Kinase Signal Transduction Pathway in Nucleus Tractus Solitarii of (mRen2)27 Transgenic Rats improves Baroreceptor Sensitivity

The phosphoinositol kinase (PI3K) pathway plays a role in the maintenance of resting mean arterial pressure (MAP) in vivo but the exact mechanisms remain unclear. Our goal was to determine whether the PI3K pathway modulates arterial baroreceptor reflex sensitivity (BRS) via actions in the solitary tract nucleus (nTS) of (mRen2)27 transgenic hypertensive rats. Male Hannover Sprague‐Dawley (HnSD) and (mRen2)27 rats (9–20 wk old) were anesthetized with urethane/chloralose. MAP was measured before and after bilateral nTS microinjections of the PI3K inhibitor wortmannin (100 nmol/L, 50nL). BRS was assessed before and after wortmannin treatment by intravenous infusion of increasing concentrations of phenylephrine to elicit changes in MAP and heart rate. NTS injections of wortmannin in (mRen2)27 rats significantly decreased in MAP (−32 ± 6 mm Hg; n = 5) for ~90 minutes. Wortmannin failed to decrease MAP in HnSD rats (−4 ± 3 mm Hg; n = 5). BRS improved following wortmannin treatment in (mRen2)27 rats (0.2 + 0.1 vs 0.7 ± 0.2 msec/mm Hg; p &lt; 0.05); however the BRS of HnSD rats decreased (2.1 ± 0.5 vs 1.2 ± 0.5 msec/mm Hg; p &lt; 0.05) following PI3K blockade. These results demonstrate that the PI3K pathway in the nTS of hypertensive rats impairs BRS and contributes to elevation of MAP. Further studies are required to determine the mechanisms responsible for the differences in direction of changes in BRS between (mRen2)27 and HnSD rats.

The differential effects of imidazoline‐like drugs on the components of baroreflex function in the nucleus of the solitary tract in anesthetized rats

Imidazoline‐like drugs (clonidine and moxonidine), agonists of I1‐imidazoline receptor (I1R) and/or α2‐adrenoceptors (α2AR), has been suggested to produce the cardiovascular inhibition in the central nervous system, which is associated with baroreflex modulation. The nucleus of the solitary tract (NTS) is a crucial region involved in cardiovascular and baroreflex regulation. This study was to comparatively detect the effects of clonidine and moxonidine on the baroreflex control of heart rate (HR) or renal sympathetic nerve activity (RSNA) in normotensive rats. In urethane and α‐chloralose anesthetized rats, bilateral microinjection of clonidine (2 nmol), a mixed agonist of I1R and α2AR, into the NTS increased the baroreflex control of HR (1.13 ± 0.1 vs. 1.79 ± 0.2 bpm/mmHg, P&lt;0.05) and RSNA (1.19 ± 0.2 vs. 1.73 ± 0.3 %/mmHg, P&lt;0.05). Bilateral NTS injection of the selective I1R agonist moxonidine (4 nmol) increased the baroreflex control of RSNA (1.22 ± 0.2 vs. 1.79 ± 0.3 %/mmHg, P&lt;0.05) without affecting the baroreflex control of HR (1.15 ± 0.1 vs. 1.18 ± 0.2 bpm/mmHg, P&gt;0.05). In conclusion, the present results demonstrate that the imidazoline‐like drug clonidine and moxonidine in the NTS differentially modulate the baroreflex components, which probably is dependent on acting receptors. (Supported by the National Natural Science Foundation of China, No. 30470636 and 30670759)

Low-Dose Local Kidney Irradiation Inhibits Progression of Experimental Crescentic Nephritis by Promoting Apoptosis

Background: Crescentic glomerulonephritis is a rapidly progressive form of nephritis and is usually resistant to therapeutic intervention. Apoptosis plays a role in the resolution of glomerulonephritis. We investigated the effects of local kidney irradiation on the progression of experimental crescentic glomerulonephritis. Methods: The following three experimental rat groups were generated: (1) Group I, sham-operated control (n = 12); (2) Group II, rats injected intravenously with rabbit anti-rat GBM antibody (nephrotoxic serum, NTS) (n = 23), and (3) Group III, a single low-dose irradiation of 0.5 Gy X-ray to both kidneys at days 6, 13, 20, and 27 after NTS injection (n = 55). Renal function and blood leukocyte count were examined in different groups of rats at various time points. Kidneys obtained at various time points were analyzed to determine the effects of radiation in experimental nephritis. Results: Radiation of the kidneys reduced the levels of blood urea nitrogen and serum creatinine compared with Group II nephritic rats of similar age (p < 0.05 or 0.001). No apparent changes in blood leukocyte counts were noted in various experimental groups. Glomerular hypercellularity, crescents, global sclerosis and tubulointerstitial damage developed gradually in Group II rats, but were decreased (p < 0.05 or 0.001) after radiation treatment. The extent of tubulointerstitial damage was also reduced, and radiation-associated histological improvements were accompanied by reduced infiltration of macrophages in the glomeruli and interstitium. The numbers of PCNA- and ED1-positive cells were reduced in the kidneys at 1 day post-irradiation, of rats irradiated at 6 and 13 days after NTS injection, compared with Group II at similar time intervals (p < 0.05). A larger numbers of TUNEL-positive cells were noted at 1 day post-irradiation in rats irradiated at 6 and 13 days after NTS injection, compared with Group II at similar time intervals (p < 0.05). Immunostaining for macrophages ED1 and TUNEL staining of serial sections of irradiated nephritic kidneys showed few ED1-positive macrophages stained for TUNEL. Overexpression of active caspases 3 and 7 was noted in irradiated kidneys, compared with the corresponding Group II rats at similar time intervals. Western blot analysis showed marked increase in active caspase 3 and active caspase 7 expression in irradiated kidneys compared with NTS injection only. A marked increase in the expression of p53 protein, which is closely related to radiation-induced apoptosis, was also observed in irradiated kidneys compared with NTS injection only. Conclusion: Our study showed that renal radiation can alter acute glomerular inflammation by inducing apoptosis of intrinsic and infiltrating cells in the kidney in a rat model of crescentic glomerulonephritis. Low-dose kidney irradiation can inhibit the progression of experimental nephritis through inducing apoptosis.

Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius

Hypocretin-1 (hcrt-1)-containing axons have been shown to have an extensive distribution within the central nervous system, although the total number of hypothalamic hcrt-1 neurons has been shown to be small. This suggests that hcrt-1 neurons may innervate central structures with similar function through collateral axonal projections. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether hypothalamic hcrt-1-containing neurons send collateral axonal projections to cardiovascular sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb) in the rat. Fluorogold- (FG) and/or rhodamine (Rd)-labeled latex microspheres were microinjected into either the NTS or Amb at sites that elicited bardycardia responses ( l-glutamate; 0.25 M; 10 nl). After a survival period of 10–15 days, the rats were sacrificed and tissue sections of the hypothalamus were processed immunohistochemically for the identification of hcrt-1-containing cell bodies. After injection of the tract-tracers into the NTS or Amb, retrogradely labeled neurons were observed within several hypothalamic regions; the paraventricular hypothalamic nucleus, lateral hypothalamic area, perifornical hypothalamic area, and posterior hypothalamus, bilaterally, but with an ipsilateral predominance. In addition, after NTS injections, retrogradely labeled neurons were found within the ipsilateral caudal arcuate nucleus. Of the total number (1107±97) of hcrt-1-immunoreactive neurons found bilaterally within the lateral and perifornical hypothalamic nuclei, 7.9±1.4% were found to be retrogradely labeled from the NTS, 16.4±1.8% from the Amb, and 3.1±0.5% from both medullary sites. Hcrt-1 neurons projecting to the NTS were found mainly in and around the perifornical hypothalamic region, with a smaller number in the caudal lateral hypothalamic area. On the other hand, those innervating the Amb were primarily observed within the caudal lateral hypothalamic area, with a smaller number in the perifornical hypothalamic area. Neurons with collateral axonal projections to NTS and Amb were observed within two specific hypothalamic areas: one group of neurons was found in the perifornical hypothalamic area, and the other was observed in the lateral hypothalamic region just dorsal to the retrochiasmatic component of the supraoptic nucleus. These data indicate that axons from hcrt-1 neurons bifurcate to innervate functionally similar cardiovascular-responsive sites in the NTS and Amb. Although the function of these hcrt-1-containing hypothalamic–medullary pathways is not known, they likely represent the anatomical substrate by which the lateral hypothalamic hcrt-1 neurons simultaneously coordinate autonomic–cardiovascular responses to different behaviors.

Nociception attenuates parasympathetic but not sympathetic baroreflex via NK1 receptors in the rat nucleus tractus solitarii.

Somatic noxious stimulation can evoke profound cardiovascular responses by altering activity in the autonomic nervous system. This noxious stimulation attenuates the cardiac vagal baroreflex, a key cardiovascular homeostatic reflex. This attenuation is mediated via NK1 receptors expressed on GABAergic interneurones within the nucleus of the solitary tract (NTS). We have investigated the effect of noxious stimulation and exogenous substance P (SP) on the sympathetic component of the baroreflex. We recorded from the sympathetic chain in a decerebrate, artificially perfused rat preparation. Noxious hindlimb pinch was without effect on the sympathetic baroreflex although the cardiac vagal baroreflex gain was decreased (56 %, P < 0.01). Bilateral NTS microinjection of SP (500 fmol) produced a similar selective attenuation of the cardiac vagal baroreflex gain (62 %, P < 0.005) without effect on the sympathetic baroreflex. Recordings from the cardiac sympathetic and vagal nerves confirmed the selectivity of the SP inhibition. Control experiments using a GABAA receptor agonist, isoguvacine, indicated that both components of the baroreflex (parasympathetic and sympathetic) could be blocked from the NTS injection site. The NTS microinjection of a NK1 antagonist (CP-99,994) in vivo attenuated the tachycardic response to hindlimb pinch. Our data suggest that noxious pinch releases SP within the NTS to selectively attenuate the cardiac vagal, but not the sympathetic, component of the baroreflex. This selective withdrawal of the cardiac vagal baroreflex seems to underlie the pinch-evoked tachycardia seen in vivo. Further, these findings confirm that baroreflex sympathetic and parasympathetic pathways diverge, and can be independently controlled, within the NTS.