Control Of Sympathetic Outflow Research Articles

Centrally Projecting Edinger-Westphal Nucleus in the Control of Sympathetic Outflow and Energy Homeostasis.

The centrally projecting Edinger-Westphal nucleus (EWcp) is a midbrain neuronal group, adjacent but segregated from the preganglionic Edinger-Westphal nucleus that projects to the ciliary ganglion. The EWcp plays a crucial role in stress responses and in maintaining energy homeostasis under conditions that require an adjustment of energy expenditure, by virtue of modulating heart rate and blood pressure, thermogenesis, food intake, and fat and glucose metabolism. This modulation is ultimately mediated by changes in the sympathetic outflow to several effector organs, including the adrenal gland, heart, kidneys, brown and white adipose tissues and pancreas, in response to environmental conditions and the animal’s energy state, providing for appropriate energy utilization. Classic neuroanatomical studies have shown that the EWcp receives inputs from forebrain regions involved in these functions and projects to presympathetic neuronal populations in the brainstem. Transneuronal tracing with pseudorabies virus has demonstrated that the EWcp is connected polysynaptically with central circuits that provide sympathetic innervation to all these effector organs that are critical for stress responses and energy homeostasis. We propose that EWcp integrates multimodal signals (stress, thermal, metabolic, endocrine, etc.) and modulates the sympathetic output simultaneously to multiple effector organs to maintain energy homeostasis under different conditions that require adjustments of energy demands.

Aerobic exercise regulates synaptic transmission and reactive oxygen species production in the paraventricular nucleus of spontaneously hypertensive rats

Aerobic exercise lowers blood pressure in patients with hypertension, but the underlying mechanisms remain incompletely understood. The hypothalamic paraventricular nucleus (PVN) plays a key role in the control of sympathetic outflow and cardiovascular tone. We examined whether chronic aerobic exercise altered synaptic transmission and reactive oxygen species (ROS) production in the PVN. In the present study, spontaneously hypertensive rats (SHRs) were subjected to exercise training for 8 weeks, five times per week, with Wistar Kyoto (WKY) rats as the cohort control. Miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were recorded from the PVN in ex vivo hypothalamic slice preparations obtained after the last training, and biomarkers of oxidative stress and physical indexes were observed. The mean frequency and amplitude, as well as the rise time and the decay time constant of mIPSCs, significantly decreased in 20-wk-old SHRs compared to WKY 20-wk-old controls. In contrast to mIPSCs, only the mean mEPSC frequency was higher, and there were no other changes in mEPSCs in comparison to the control group. SHRs exhibited higher ROS, 8-OHdG, and MDA; and lower SOD1, SOD2, CAT, Ogg1, and SOD and CAT activity in the PVN. These SHRs also had a significant increase in heart rate, blood pressure and sympathetic nerve activity, and higher levels of norepinephrine (NE). Exercise training ameliorated all these abnormalities, resulting in an increase in the mean frequency, amplitude and kinetics of mIPSCs, accompanied by a decrease in the mean frequency of mEPSCs in the PVN. This study demonstrates that moderate intensity, high frequency exercise training induces a selective enhancement of inhibitory synaptic transmission in the PVN, which may dampen sympathetic activity and reduce blood pressure in hypertension. These changes may be due to antioxidant-related adaptations in the PVNs of SHRs.

Muscle sympathetic nerve activity and volume-regulating factors in healthy pregnant and nonpregnant women.

Healthy, normotensive human pregnancies are associated with striking increases in both plasma volume and vascular sympathetic nerve activity (SNA). In nonpregnant humans, volume-regulatory factors including plasma osmolality, vasopressin, and the renin-angiotensin-aldosterone system have important modulatory effects on control of sympathetic outflow. We hypothesized that pregnancy would be associated with changes in the relationships between SNA (measured as muscle SNA) and volume-regulating factors, including plasma osmolality, plasma renin activity, and arginine vasopressin (AVP). We studied 46 healthy, normotensive young women (23 pregnant and 23 nonpregnant). We measured SNA, arterial pressure, plasma osmolality, plasma renin activity, AVP, and other volume-regulatory factors in resting, semirecumbent posture. Pregnant women had significantly higher resting SNA (38 ± 12 vs. 23 ± 6 bursts/min in nonpregnant women), lower osmolality, and higher plasma renin activity and aldosterone (all P < 0.05). Group mean values for AVP were not different between groups [4.64 ± 2.57 (nonpregnant) vs. 5.17 ± 2.03 (pregnant), P > 0.05]. However, regression analysis detected a significant relationship between individual values for SNA and AVP in pregnant (r = 0.71, P < 0.05) but not nonpregnant women (r = 0.04). No relationships were found for other variables. These data suggest that the link between AVP release and resting SNA becomes stronger in pregnancy, which may contribute importantly to blood pressure regulation in healthy women during pregnancy.NEW & NOTEWORTHY Sympathetic nerve activity and blood volume are both elevated during pregnancy, but blood pressure is usually normal. Here, we identified a relationship between vasopressin and sympathetic nerve activity in pregnant but not nonpregnant women. This may provide mechanistic insights into blood pressure regulation in normal pregnancy and in pregnancy-related hypertension.

GABA in Paraventricular Nucleus Regulates Adipose Afferent Reflex in Rats.

BackgroundChemical stimulation of white adipose tissue (WAT) induces adipose afferent reflex (AAR), and thereby causes a general sympathetic activation. Paraventricular nucleus (PVN) is important in control of sympathetic outflow. This study was designed to investigate the role of γ-aminobutyric acid (GABA) in PVN in regulating the AAR.Methodology/Principal FindingsExperiments were carried out in anesthetized rats. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. AAR was evaluated by the RSNA and MAP responses to electrical stimulation of the right epididymal WAT (eWAT) afferent nerve. Electrical stimulation of eWAT afferent nerve increase RSNA. Bilateral microinjection of the GABAA receptor agonist isoguvacine or the GABAB receptor agonist baclofen attenuated the AAR. The effect of isoguvacine on the AAR was greater than that of baclofen. The GABAA receptor antagonist gabazine enhanced the AAR, while the GABAB receptor antagonist CGP-35348 had no significant effect on the AAR. Bilateral PVN microinjection of vigabatrin, a selective GABA-transaminase inhibitor, to increase endogenous GABA levels in the PVN abolished the AAR. The inhibitory effect of vigabatrin on the AAR was attenuated by the pretreatment with gabazine or CGP-35348. Pretreatment with combined gabazine and CGP-35348 abolished the effects of vigabatrin.ConclusionsActivation of GABAA or GABAB receptors in the PVN inhibits the AAR. Blockade of GABAA receptors in the PVN enhances the AAR. Endogenous GABA in the PVN plays an important role in regulating the AAR.

Norepinephrine (NE) Increases Production of Superoxide (O2• ‐) in Cultured Peripheral Blood Mononuclear Cells (PBMCs) and Splenocytes Isolated from Rats

Physiological/pathophysiological states with increased sympathetic nerve activity (SNA) are associated with oxidative stress. It is known that increased O2• ‐ in specific brain regions results in increased efferent SNA. Recently we found that NE increased intracellular O2• ‐ production (Dihydroethidium, DHE) and mRNA for NADPH oxidase (NOX‐2) subunits in cultured PBMCs from humans suggesting elevated SNA may increase systemic O2• ‐ production. Current experiments tested whether the rat is a good experimental model of humans to further study the interaction between sympathoexcitation and PBMC‐derived O2• ‐. Primary PBMCs and splenocytes were isolated from male rats and incubated at 37°C for 36 hr with either vehicle (Veh), 50 pcg/ml, 50 ng/ml, or 50 µg/ml NE. Similar to human PBMCs, relative to Veh, NE increased intracellular O2• ‐ production in rat PBMCs (50 µg/ml NE = 11± 5 fold; P&lt; 0.05) and tended to increase mRNA for NOX‐2 subunits (50 ng/ml NE: gp 91phox = 1.8 ± 0.4; p22phox = 1.6± 0.5). NE also increased O2• ‐ in rat splenocytes (50 µg/ml NE = 11± 6 fold; P&lt; 0.05). Thus, results in rat PBMCs and splenocytes are similar to human PBMCs, and are consistent with a positive feedback system whereby increased NE promotes increased O2• ‐ in circulating PBMCs which in turn could activate CNS sites to promote further increases in SNA. Using the rat as a model, future experiments will evaluate a potential role of circulating PBMCs on activation of brain regions involved in the control of sympathetic outflow. R01 HL091164

Central mechanisms for exercise training-induced reduction in sympatho-excitation in chronic heart failure.

The control of sympathetic outflow in the chronic heart failure (CHF) state is markedly abnormal. Patients with heart failure present with increased plasma norepinephrine and increased sympathetic nerve activity. The mechanism for this sympatho-excitation is multiple and varied. Both depression in negative feedback sensory control mechanisms and augmentation of excitatory reflexes contribute to this sympatho-excitation. These include the arterial baroreflex, cardiac reflexes, arterial chemoreflexes and cardiac sympathetic afferent reflexes. In addition, abnormalities in central signaling in autonomic pathways have been implicated in the sympatho-excitatory process in CHF. These mechanisms include increases in central Angiotensin II and the Type 1 receptor, increased in reactive oxygen stress, upregulation in glutamate signaling and NR1 (N-methyl-D-aspartate subtype 1) receptors and others. Exercise training in the CHF state has been shown to reduce sympathetic outflow and result in increased survival and reduced cardiac events. Exercise training has been shown to reduce central Angiotensin II signaling including the Type 1 receptor and reduce oxidative stress by lowering the expression of many of the subunits of NADPH oxidase. In addition, there are profound effects on the central generation of nitric oxide and nitric oxide synthase in sympatho-regulatory areas of the brain. Recent studies have pointed to the balance between Angiotensin Converting Enzyme (ACE) and ACE2, translating into Angiotensin II and Angiotensin 1-7 as important regulators of sympathetic outflow. These enzymes appear to be normalized following exercise training in CHF. Understanding the precise molecular mechanisms by which exercise training is sympatho-inhibitory will uncover new targets for therapy.

Inhibition of endoplasmic reticulum function in PVN by thapsigargin increases neuronal excitability and sympathetic nerve activity (1125.4)

Control of sympathetic outflow by paraventricular nucleus (PVN) is partially mediated through a monosynaptic axon projecting to the rostral ventrolateral medulla (PVN‐RVLM). Here we investigated the role of thapsigargin (TG), the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor, in regulating the excitability of PVN‐RVLM neurons under brain slice preparation. In current‐clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater (P &lt; 0.05) in the presence of TG (0.5 µM) (31 ± 3 Hz, n=9). The effect of ER Ca2+ store depletion in regulating renal SNA (RSNA) and mean arterial pressure (MAP) was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased RSNA and MAP in a dose‐dependent manner. Maximum increases in RSNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre‐treatment with CyPPA (5 nmol/100 nl), a small conductance, Ca2+ activated K (SK) channel activator (n=3), attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for RSNA and 4 ± 7 mmHg for MAP). PVN CyPPA did not significantly alter baseline RSNA and MAP. Our data indicate that inhibition of ER function with TG enhances in vitro excitability of PVN‐RVLM neurons and increases in vivo RSNA and MAP. The effect of TG on the excitability of PVN neurons and SNA through ER Ca2+ store depletion may involve reduced SK channel activity.Grant Funding Source: Supported by AHA10SDG2640130

Abstract 437: Inhibition of Endoplasmic Reticulum Function in Pre-sympathetic PVN Neurons by Thapsigargin Increased Neuronal Excitability and Sympathetic Nerve Activity

Control of sympathetic outflow by pre-sympathetic paraventricular nucleus (PVN) neurons is partially mediated through a monosynaptic pathway that innervates the rostral ventrolateral medulla (RVLM). Here we investigated the role of the thapsigargin (TG), the endoplasmic reticulum (ER) Ca 2+ ATPase inhibitor, in regulating the in vitro excitability of PVN neurons projecting to RVLM (PVN-RVLM) under brain slice preparation. In current-clamp recordings, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22 ± 2 Hz (n=7) and was significantly greater ( P &lt; 0.05) in the presence of TG (0.5 μ M) (32 ± 4 Hz, n=5). Next, the effect of Ca 2+ store depletion from the ER in regulating SNA and blood pressure was assessed by bilateral PVN microinjection of TG in anesthetized rats. PVN injection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased the renal sympathetic nerve activity (SNA) and mean arterial pressure (MAP) in a dose-dependent manner. Maximum increase in renal SNA and MAP elicited by PVN TG (0.75 nmol/100nl; n=3) were 105 ± 31% and 18 ± 11 mmHg respectively. Pre-treatment of CyPPA (5 nmol/100 nl), a SK channel activator/opener (n=3), remarkably attenuated the PVN TG evoked sympathoexcitatory and pressor responses (7 ± 7% for renal SNA and 4 ± 7 mmHg for MAP). PVN injection of CyPPA did not significantly alter the baseline of renal SNA and MAP. Our data indicate that inhibition of ER function by TG contributes to the enhanced in vitro excitability of PVN-RVLM neurons and increased in vivo renal SNA and MAP. The effect of TG on the excitability of pre-sympathetic PVN neurons and sympathetic outflow through the Ca 2+ store depletion from the ER may involve the down-regulation of SK channel function. Support: NSDP2010008 (JZ) and AHA10SDG2640130 (CQH).

The neural interaction between the arterial baroreflex and muscle metaboreflex is preserved in older men

Sympathetic baroreflex sensitivity is increased during selective activation of the skeletal muscle metaboreflex with postexercise ischaemia (PEI) in young adults. However, to date, there are no data demonstrating this neural interaction between the arterial baroreflex and the muscle metaboreflex in healthy older adults. Therefore, the goal of the present study was to examine the influence of healthy ageing on the metabolic component of the exercise pressor reflex and its interaction with the arterial baroreflex in the control of sympathetic outflow. Postexercise ischaemia following static hand grip performed at 30% maximal voluntary contraction was used to isolate muscle metaboreflex activation in young [n = 10; 24 ± 1 years old; resting blood pressure (BP) 116 ± 3/64 ± 3 mmHg] and older men (n = 9; 59 ± 2 years old; resting BP 120 ± 2/77 ± 2 mmHg). Arterial BP (Finometer) and muscle sympathetic nerve activity (MSNA) were measured continuously. Weighted linear regression analysis between MSNA and diastolic BP was used to estimate arterial baroreflex MSNA gain. There were no age-related differences in the increase in mean BP (young, Δ14 ± 3 mmHg versus older, Δ15 ± 2 mmHg; P > 0.05) or MSNA burst frequency (young, Δ11 ± 2 bursts min(-1) versus older, Δ9 ± 1 bursts min(-1); P > 0.05) during PEI. Likewise, the gain of arterial baroreflex control of total MSNA increased to a similar extent in both groups during PEI (young, -4.2 ± 0.9 baseline versus -6.3 ± 1.1 PEI a.u. beat(-1) mmHg(-1); and older, -3.7 ± 1.1 baseline versus -6.7 ± 1.4 PEI a.u. beat(-1) mmHg(-1); P < 0.05 for both). Collectively, these findings indicate that the neural interaction between the arterial baroreflex and the skeletal muscle metaboreflex in the regulation of MSNA is preserved in healthy ageing.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P(CO)(2)) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO(2) from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO(2) exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO(2) causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO(2)-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

Neural control of the circulation

The purpose of this brief review is to highlight key concepts about the neural control of the circulation that graduate and medical students should be expected to incorporate into their general knowledge of human physiology. The focus is largely on the sympathetic nerves, which have a dominant role in cardiovascular control due to their effects to increase cardiac rate and contractility, cause constriction of arteries and veins, cause release of adrenal catecholamines, and activate the renin-angiotensin-aldosterone system. These effects, as well as the control of sympathetic outflow by the vasomotor center in the medulla and the importance of sensory feedback in the form of peripheral reflexes, especially the baroreflexes, are discussed in the context of cardiovascular regulation.

Increased Group I Metabotropic Glutamate Receptor Activity in Paraventricular Nucleus Contributes to Elevated Sympathetic Vasomotor Tone in Hypertension

Enhanced glutamatergic input in the paraventricular nucleus (PVN) contributes to elevated sympathetic outflow in hypertension. We determined the role of group I metabotropic glutamate receptors (mGluRs) in the PVN in the control of sympathetic outflow in spontaneously hypertensive rats (SHR). Lumbar sympathetic nerve activity (LSNA), arterial blood pressure (Bp), and heart rate (HR) were recorded from anesthetized SHR and WKY rats. Bilateral microinjection of MPEP (1–10 nmol), a mGluR5 antagonist, or LY367385 (10–100 nmol), a mGluR1 antagonist, into the PVN dose‐dependently decreased LSNA and Bp in SHR but not in WKY. MPEP‐induced decreases in LSNA and Bp were greater than that produced by LY367385 in SHR. Furthermore, microinjection of group I mGluR agonist DHPG (10–100 nmol) caused dose‐dependent increase in LSNA and Bp in both WKY and SHR. DHPG‐induced responses were significantly attenuated by MPEP or LY367385 and abolished by a combination of MPEP and LY367385 in both WKY and SHR. In addition, prior injection of NMDA receptor antagonist AP5 (1.4 nmol) into the PVN decreased LSNA and Bp in SHR and attenuated DHPG‐induced sympathoexcitation in both WKY and SHR. Thus, our study suggests that elevated sympathetic outflow is maintained by activation of group I mGluR receptors in the PVN in SHR. Increased group I mGluR receptor activity contributes to increased glutamatergic input via NMDA receptor in hypertension.

S7. Central cardiovascular control: Plasticity in response to normal physiological challenges: S7.1 Pregnancy: CNS plasticity in control of sympathetic outflow in rats

Normal pregnancy is characterized by a ~40% increase in blood volume and cardiac output, mild tachycardia, and decreased arterial blood pressure due to a substantial decrease in total peripheral resistance (TPR). Increased susceptibility to orthostatic and hemorrhagic hypotension is significant problems in term pregnant women and animals, and a number of studies suggest that arterial baroreflex compensatory responses are suppressed in pregnancy. In near term pregnant rats and rabbits, maximum gain for arterial baroreflex control of renal sympathetic nerve activity (RSNA) is decreased, largely due to an attenuated ability to increase RSNA in response to a hypotensive challenge.

Input–output relationships of a somatosympathetic reflex in human spinal injury

Spinal cord injury results in loss of supraspinal control of sympathetic outflow, yet preservation of spinal reflexes. Given the importance of reflex activation of sympathetic vasoconstrictor neurones to the generation of autonomic dysreflexia, we assessed the input-output relationship of the spinal somatosympathetic reflex induced by electrical activation of cutaneous afferents over the lower abdominal wall. In 13 spinal cord-injured subjects (C4-T10) we tested the hypothesis that the magnitude and duration of the vasoconstriction is directly related to the magnitude and duration of the stimulus train. Cutaneous vasoconstriction was measured with photoelectric plethysmography over a finger and toe; continuous blood pressure was recorded by radial artery tonometry, heart rate by ECG chest electrodes and sweat release by skin conductance. Four sets of trains of cutaneous electrical stimuli (20 Hz 1 s, 20 Hz 20 s, 20 Hz 1 s alternating on-and-off for 20 s and 1 Hz 20 s) were applied to the abdominal wall (10 mA) at 2-min intervals. Nine subjects showed vasoconstrictor responses to the stimulus trains. On average, both the magnitude and duration of the responses were similar irrespective of the type of stimulus train. We conclude that there is a non-linear relationship between somatic inputs and sympathetic vasoconstrictor outputs, and argue that a sustained vasoconstriction need not imply continuous sensory input to the spinal cord.

TRPV1‐expressing Afferents Innervate the Aorta and Contribute to Baroreflex Control of Cardiovascular Function

The arterial baroreflex is important in the regulation of blood pressure and sympathetic outflow. The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary sensory neurons including baroreceptive neurons. Here, we tested the hypothesis that TRPV1‐expressing afferents participate in the control of sympathetic outflow. We used resiniferatoxin (RTX), an ultrapotent analogue of capsaicin, to remove TRPV1‐expressing neurons and afferent fibers in rats. Enhanced immunofluorescent staining showed that TRPV1‐immunoreactivities were present on the nerve endings in the ascending aorta and aortic arch, nodose ganglion (NG) neurons, and afferent nerve endings and neurons in the nucleus solitary tract (NTS). RTX treatment removed TRPV1 immunoreactivities in the aorta, NG neurons, and NTS. Renal sympathetic nerve activity (RSNA), arterial blood pressure (ABP), and heart rate (HR) were recorded in anesthetized rats receiving RTX treatment. The baroreflex was triggered by lowering and raising blood pressure via intravenous infusion of nitroglycerin and phenylephrine, respectively. The baroreflex control of RSNA and HR were impaired in high blood pressure range in RTX‐treated rats. Furthermore, the maximum gain of the baroreflex control of RSNA and HR were significantly decreased in RTX‐treated rats. Furthermore, blocking TRPV1 receptors with Iodo‐RTX blunted the baroreflex control of RSNA and HR and decreased the maximum gain of baroreflex function curves in high blood pressure range. These data provide important new evidence that TRPV1 receptors are expressed in afferent nerves and neurons along the baroreflex pathway. TRPV1‐expressing neurons contributes to the baroreflex control of the cardiovascular function.

Dynamic arterial baroreflex function during high intensity exercise in humans: insights into sympathetic control

Dynamic arterial baroreflex function during high intensity exercise in humans: insights into sympathetic control

Increased nitric oxide synthase activity and expression in the hypothalamus of hindlimb unloaded rats

Upon return from spaceflight or resumption of normal posture after bed rest, individuals often exhibit cardiovascular deconditioning. Although the mechanisms responsible for cardiovascular deconditioning have yet to be fully elucidated, alterations within the central nervous system have been postulated to be involved. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important brain regions in control of sympathetic outflow and body fluid homeostasis. Nitric oxide (NO) modulates the activity of PVN and SON neurons, and alterations in NO transmission within these brain regions may contribute to symptoms of cardiovascular deconditioning. The purpose of the present study was to examine nitric oxide synthase (NOS) activity and expression in the PVN and SON of control and hindlimb unloaded (HU) rats, an animal model of cardiovascular deconditioning. The number of neurons exhibiting NOS activity as assessed by NADPH-diaphorase staining was significantly greater in the PVN but not SON of HU rats. Western blot analysis revealed that neuronal NOS (nNOS) but not endothelial NOS (eNOS) protein expression was higher in the PVN of HU rats. In the SON, there was a strong trend for an increase in nNOS ( p = 0.052) and a significant increase in eNOS expression in HU rats. Our results suggest that increased nNOS in the PVN contributes to autonomic and humoral alterations following cardiovascular deconditioning. In contrast, the functional significance of increases in nNOS and eNOS protein in the SON may be related to alterations in vasopressin release observed previously in HU rats.

Laser Capture Microscopy and real time RT‐PCR: Phenotyping spinally projecting brainstem and forebrain neurons

These experiments demonstrate the use of laser capture microscopy (LCM) and real time RT-PCR to evaluate neurotransmitters, neuromodulators and receptor expression in spinally projecting neurons in the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus of the hypothalamus (PVN). Fluororuby (1%, 90 nl) was microinjected into the intermediolateral cell column of the thoracic spinal cord (T1 – T2) 7 days prior to harvesting brains. Brains were frozen, 10 μ sections obtained from forebrain and brainstem, and dehydrated. Retrogradely labeled neurons were visualized and microdissected from the RVLM and PVN using Capsure MacroLCM caps and a PixCell IIe laser capture microscope (20X; Spot size = 7.5 μ). RNA was extracted and cDNA synthesized using standard procedures. Real time RT-PCR (Cepheid Smart Cycler II) using SYBR green was performed for mRNAs of GAPDH (house keeping gene), GABAA receptor subunits and nNOS. Standards for target PCR products had been previously cloned, isolated, purified, and quantified with spectrophotometry. Serial dilutions of the appropriate standards were used to quantitate amount of product. Caps with ~15–25 cells yielded 24 μl of cDNA and quantifiable amounts of GAPDH, GABAA α1 & δ subunit, and nNOS in 1 μl aliquots. Values are ratio to GAPDH from the same cap (pmol/nmol GAPDH). [RVLM: α1= 251.5; delta; = 223.7; nNOS= 34.6; PVN: α1= 192.8; delta; = 66.4; nNOS= 98.9]. It is predicted that 6-10 mRNA’s can be determined from each cap of pooled cells. Thus, LCM and real time RT-PCR will allow for detailed phenotyping of neurons involved in control of sympathetic outflow. (NIH HL36245)

Expression of cardiovascular and behavioural components of conditioned fear to context in T4 spinally transected rats

A spinal cord transection at the fourth thoracic level (T4) results in paraplegia. It also removes supraspinal control of sympathetic outflow to most viscera and their blood vessels but spares the heart. We studied the effects of such a transection on the expression of the conditioned fear response to context, which includes freezing, 22 kHz ultrasonic vocalisations, a marked pressor response and a slowly rising tachycardia. Rats implanted with radiotelemetric probes were fear conditioned, tested, then transected at T4 and finally re-tested 4 weeks after transection. Baseline blood pressure in transected animals was the same as in intact animals but baseline heart rate was 127 bpm higher. There were clear signs of fear in the transected animals: although freezing occurred in the upper part of the body only, there was a 3 fold increase in the number of ultrasonic vocalisations, most probably due to paralysis of abdominal muscles that made expirations shorter and therefore more frequent. The pressor response of fear was initially the same as in intact animals but controls revealed that this was due to handling during transfer to the aversive context. The rest of the pressor response was markedly reduced (70%) confirming that it depends in large part on a sympathetically mediated increase in vascular resistance in the lower part of the body. The cardiac response was characterized by an initial bradycardia followed by a marked tachycardia, which is consistent with a baroreceptor-mediated reflex response to the altered pressor changes. Finally, none of these changes was observed when the same experiment was repeated in sham transected animals. Thus, the pressor response of fear is in large part mediated by the thoracic cord below T4 and the baroreflex is not inhibited but maintained during conditioned fear.

Angiotensin II Stimulates Spinally Projecting Paraventricular Neurons through Presynaptic Disinhibition

Paraventricular nucleus (PVN) neurons that project to the spinal cord are important in the control of sympathetic outflow. Angiotensin II (Ang II) can stimulate PVN neurons, but its cellular mechanisms are not clear. In this study, we determined the effect of Ang II on the excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Whole-cell patch-clamp recordings were performed on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Immunocytochemistry labeling revealed that the immunoreactivity of angiotensin type 1 (AT1) receptors was colocalized with a presynaptic marker, synaptophysin, in the PVN. Application of 0.1-5 microm Ang II significantly decreased the amplitude of evoked GABAergic IPSCs in a concentration-dependent manner. Also, Ang II decreased the frequency of miniature IPSCs from 2.56 +/- 0.45 to 1.05 +/- 0.20 Hz (p < 0.05; n = 12), without affecting the amplitude and the decay time constant. The effect of Ang II on miniature IPSCs was blocked by losartan but not PD123319. However, Ang II had no effect on the evoked glutamatergic EPSCs and did not alter the frequency and amplitude of miniature EPSCs at concentrations that attenuated IPSCs. Furthermore, Ang II increased the firing rate of PVN neurons from 3.75 +/- 0.36 to 7.89 +/- 0.85 Hz (p < 0.05; n = 9), and such an effect was abolished by losartan. In addition, Ang II failed to excite PVN neurons in the presence of bicuculline. Thus, this study provides substantial new evidence that Ang II excites spinally projecting PVN neurons by attenuation of GABAergic synaptic inputs through activation of presynaptic AT1 receptors.