The left ventricle increases contractility in response to baroreceptor unloading, which is sympathetically-mediated in the anesthetized rat.

BACKGROUND: Contemporary discussion of the baroreflex includes the efferent vascular-sympathetic and cardio-vagal arms. Since sympathetic post-ganglionic neurons also innervate the left ventricle (LV), it is oft-assumed that the LV produces a sympathetically-mediated increase in contractility during baroreceptor unloading, but this has not been characterized using a load-independent index of contractility. We aimed to determine a) whether LV contractility increases in response to baroreceptor unloading, and b) whether such increases are mediated via the sympathetic or parasympathetic arm of the autonomic nervous system. METHODS: Ten male Wistar rats were anesthetized (urethane) and instrumented with arterial and LV pressure-volume catheters to measure mean arterial pressure (MAP) and load-independent LV contractility [maximal rate of increase in pressure adjusted to end-diastolic volume (PAdP/dtmax)], respectively. Rats were placed in a servo-controlled lower-body negative pressure (LBNP) chamber to reduce MAP by 10% for 60s to mechanically unload baroreceptors under control conditions. LBNP was repeated in each animal following infusions of cardiac autonomic blockers using esmolol (sympathetic), atropine (parasympathetic), and esmolol+atropine. RESULTS: Under control conditions, PAdP/dtmax increased during baroreceptor unloading (26±6 vs. 31±9 mmHg·s-1·µL-1, p=0.031). During esmolol, there was no increase in LV contractility during baroreceptor unloading (11±2 vs. 12±2, p=0.125); however, during atropine, there was an increase in LV contractility during baroreceptor unloading (26±6 vs. 31±9, p=0.019). During combined esmolol and atropine, there was a small increase in contractility vs control (13±3 vs. 15±4, p=0.046). CONCLUSION: Our results demonstrate that, in anesthetized rats, LV contractility increases in response to baroreceptor unloading, which is largely sympathetically mediated.

Influence of sex and sedentary conditions on sympathetic burst characteristics in prepubertal, postpubertal, and young adult rats.

Recent evidence indicates that sex-based differences in cardiovascular disease (CVD) begin early in life, particularly when associated with risk factors such as a sedentary lifestyle. CVD is associated with elevated sympathetic nerve activity (SNA), quantified as increased SNA burst activity in humans. Whether burst characteristics are influenced by sex or sedentary conditions at younger ages is unknown. The purpose of our study is to compare SNA bursts in active and sedentary female and male rats at ages including prepuberty and young adulthood. We hypothesized that burst characteristics and blood pressure are higher under sedentary conditions and lower in female rats compared with males. We analyzed splanchnic SNA (SpSNA) recordings from Inactin-anesthetized male and female rats at 4-, 8-, and 16-wk of age. Physically active and sedentary rats were each housed in separate, environmentally controlled chambers where physically active rats had free access to an in-cage running wheel. Sympathetic bursts were obtained by rectifying and integrating the raw SpSNA signal. Burst frequency, burst height, and burst width were calculated using the Peak Parameters extension in LabChart. Our results showed that sedentary conditions produced a greater burst width in 8- and 16-wk-old rats compared with 4-wk-old rats in both males and females (P < 0.001 for both). Burst frequency and incidence were both higher in 16-wk-old males compared with 16-wk-old females (P < 0.001 for both). Our results suggest that there are sedentary lifestyle- and sex-related mechanisms that impact sympathetic regulation of blood pressure at ages that range from prepuberty into young adulthood.NEW & NOTEWORTHY The mechanisms of decreased incidence of cardiovascular disease (CVD) in reproductive-age women compared with age-matched men are unknown. The strong association between elevated sympathetic activity and CVD led us to characterize splanchnic sympathetic bursts in female and male rats. Prepubescent males and females exhibited narrower sympathetic bursts, whereas young adult males had higher resting burst frequency compared with age-matched females. Sex-based regulation of sympathetic activity suggests a need for sex-dependent therapeutic strategies to combat CVD.

Revisiting differential control of sympathetic outflow by the rostral ventrolateral medulla.

The rostral ventrolateral medulla (RVLM) is an important brain region involved in both resting and reflex regulation of the sympathetic nervous system. Anatomical evidence suggests that as a bilateral structure, each RVLM innervates sympathetic preganglionic neurons on both sides of the spinal cord. However, the functional importance of ipsilateral versus contralateral projections from the RVLM is lacking. Similarly, during hypotension, the RVLM is believed to rely primarily on withdrawal of tonic gamma aminobutyric acid (GABA) inhibition to increase sympathetic outflow but whether GABA withdrawal mediates increased activity of functionally different sympathetic nerves is unknown. We sought to test the hypothesis that activation of the ipsilateral versus contralateral RVLM produces differential increases in splanchnic versus adrenal sympathetic nerve activities, as representative examples of functionally different sympathetic nerves. We also tested whether GABA withdrawal is responsible for hypotension-induced increases in splanchnic and adrenal sympathetic nerve activity. To test our hypothesis, we measured splanchnic and adrenal sympathetic nerve activity simultaneously in Inactin-anesthetized, male Sprague-Dawley rats during ipsilateral or contralateral glutamatergic activation of the RVLM. We also produced hypotension (sodium nitroprusside, i.v.) before and after bilateral blockade of GABAA receptors in the RVLM (bicuculline, 5 mM 90 nL). Glutamate (100 mM, 30 nL) injected into the ipsilateral or contralateral RVLM produced equivalent increases in splanchnic sympathetic nerve activity, but increased adrenal sympathetic nerve activity by more than double with ipsilateral injections versus contralateral injections (p < 0.05; n = 6). In response to hypotension, increases in adrenal sympathetic nerve activity were similar after bicuculline (p > 0.05), but splanchnic sympathetic nerve activity responses were eliminated (p < 0.05; n = 5). These results provide the first functional evidence that the RVLM has predominantly ipsilateral innervation of adrenal nerves. In addition, baroreflex-mediated increases in splanchnic but not adrenal sympathetic nerve activity are mediated by GABAA receptors in the RVLM. Our studies provide a deeper understanding of neural control of sympathetic regulation and insight towards novel treatments for cardiovascular disease involving sympathetic nervous system dysregulation.

Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla.

A sedentary lifestyle is the top preventable cause of death and accounts for substantial socioeconomic costs to society. The rostral ventrolateral medulla regulates blood pressure under normal and pathophysiological states, and demonstrates inactivity-related structural and functional neuroplasticity, which is subregionally specific. The purpose of this study was to examine pro- and mature forms of brain-derived neurotrophic factor (BDNF) and their respective receptors in the male rat rostral ventrolateral medulla (RVLM) and its rostral extension following sedentary vs. active (running wheels) conditions (10-12weeks). We used subregionally specific Western blotting to determine that the mature form of BDNF and its ratio to its pro-form were lower in more caudal subregions of the rostral ventrolateral medulla of sedentary rats but higher in the rostral extension when both were compared to active rats. The full-length form of the tropomyosin receptor kinase B receptor and the non-glycosylated form of the 75 kilodalton neurotrophin receptor were lower in sedentary compared to active rats. The rostrocaudal patterns of expression of the mature form of BDNF and the full-length form of the tropomyosin receptor kinase B receptor were remarkably similar to the subregionally specific patterns of enhanced dendritic branching, neuronal activity, and glutamate-mediated increases in sympathetic nerve activity observed in previous studies performed in sedentary rats. Our studies suggest signaling pathways related to BDNF within subregions of both the rostral ventrolateral medulla and its rostral extension contribute to cardiovascular disease and premature death related to a sedentary lifestyle.

Sex-dependent development of enhanced sympathoexcitation in sedentary versus physically active rats.

KEY POINTS: The rostral ventrolateral medulla (RVLM) may contribute to sex-based differences in cardiovascular disease (CVD) based on overactivation of the sympathetic nervous system observed in sedentary male rats; however, the added influence of the reproductive cycle in females is currently unknown. To our knowledge this is the first study to demonstrate greater increases in sympathetic nerve activity in response to direct activation of the RVLM in female versus male rats prior to the onset of the reproductive cycle, which persisted after the onset of the reproductive cycle. Lower resting blood pressures in females also suggest peripheral adaptations contribute to sex-based differences in CVD. Sedentary versus physically active conditions appear to promote higher resting sympathetic outflow independent of age and sex. Our results demonstrate the importance of examining sedentary conditions in the context of sex differences and the reproductive cycle in contributing to sympathetic overactivity associated with cardiovascular disease. ABSTRACT: Female reproductive hormones are considered cardioprotective based on higher risks of cardiovascular disease (CVD) in post- versus pre-menopausal women. Similarly, based on epidemiological studies, a sedentary lifestyle is also a major risk factor for CVD. The mechanisms by which sedentary conditions contribute to CVD, and their influences in the presence and absence of female reproductive hormones are unknown. We hypothesized that sexually immature male and female rats would have similar centrally mediated regulation of blood pressure, but upon sexual maturation, female rats would have lower resting blood pressure and centrally-mediated sympathoexcitation compared to age-matched males. We also predicted resting sympathetic activity would increase upon exposure to sedentary versus active conditions (voluntary wheel running) in males but not in females. We recorded splanchnic sympathetic nerve activity (SSNA) and blood pressure in 4-, 8- and 16-week-old male and female rats under Inactin anaesthesia before and during microinjections of glutamate (1-100 mM) into the rostral ventrolateral medulla (RVLM). Four-week-old female rats had lower resting blood pressure and greater sympathoexcitation following activation of the RVLM, as did 8- and 16-week-old female rats, independent of age or activity condition. Sedentary animals had higher baseline SSNA compared to active animals, independent of sex or age. Our results reveal a complex influence of the interactions between the female reproductive cycle and sedentary conditions. They also demonstrate the importance of examining sedentary conditions in the context of sex- and female reproductive cycle-dependent incidences of cardiovascular disease.

Neuroplasticity in N-methyl-d-aspartic acid receptor signaling in subregions of the rat rostral ventrolateral medulla following sedentary versus physically active conditions.

The rostral ventrolateral medulla (RVLM) is a brain region involved in normal regulation of the cardiovascular system and heightened sympathoexcitatory states of cardiovascular disease (CVD). Among major risk factors for CVD, sedentary lifestyles contribute to higher mortality than other modifiable risk factors. Previous studies suggest excessive glutamatergic excitation of presympathetic neurons in the RVLM occurs in sedentary animals. Therefore, the purpose of this study was to examine neuroplasticity in the glutamatergic system in the RVLM of sedentary and physically active rats. We hypothesized that relative to active rats, sedentary rats would exhibit higher expression of glutamate N-methyl-d-aspartic acid receptor subunits (GluN), phosphoGluN1, and the excitatory scaffold protein postsynaptic density 95 (PSD95), while achieving higher glutamate levels. Male Sprague-Dawley rats (4 weeks old) were divided into sedentary and active (running wheel) conditions for 10-12 weeks. We used retrograde tracing/triple-labeling techniques, western blotting, and magnetic resonance spectroscopy. We report in sedentary versus physically active rats: 1) fewer bulbospinal non-C1 neurons positive for GluN1, 2) significantly higher expression of GluN1 and GluN2B but lower levels of phosphoGluN1 (pSer896) and PSD95, and 3) higher levels of glutamate in the RVLM. Higher GluN expression is consistent with enhanced sympathoexcitation in sedentary animals; however, a more complex neuroplasticity occurs within subregions of the ventrolateral medulla. Our results in rodents may also indicate that alterations in glutamatergic excitation of the RVLM contribute to the increased incidence of CVD in humans who lead sedentary lifestyles. Thus, there is a strong need to further pursue mechanisms of inactivity-related neuroplasticity in the RVLM.

Compliant Titin Isoform Content Is Reduced in Left Ventricles of Sedentary Versus Active Rats.

A sedentary lifestyle is associated with increased cardiovascular risk factors and reduced cardiac compliance when compared to a lifestyle that includes exercise training. Exercise training increases cardiac compliance in humans, but the mechanisms underlying this improvement are unknown. A major determinant of cardiac compliance is the compliance of the giant elastic protein titin. Experimentally reducing titin compliance in animal models reduces exercise tolerance, but it is not known whether sedentary versus chronic exercise conditions cause differences in titin isoform content. We hypothesized that sedentary conditions would be associated with a reduction in the content of the longer, more compliant N2BA isoform relative to the stiffer N2B isoform (yielding a reduced N2BA:N2B ratio) compared to age-matched exercising controls. We obtained left ventricles from 16-week old rats housed for 12 weeks in standard (sedentary) or voluntary running wheel (exercised) housing. The N2BA:N2B ratio was decreased in the hearts of sedentary versus active rats (p = 0.041). Gene expression of a titin mRNA splicing factor, RNA Binding Motif 20 protein (RBM20), correlated negatively with N2BA:N2B ratios (p = 0.006, r = -0.449), but was not different between groups, suggesting that RBM20 may be regulated post-transcriptionally. Total phosphorylation of cardiac titin was not different between the active and sedentary groups. This study is the first to demonstrate that sedentary rats exhibit reduced cardiac titin N2BA:N2B isoform ratios, which implies reduced cardiac compliance. These data suggest that a lack of exercise (running wheel) reduces cardiac compliance and that exercise itself increases cardiac compliance.

Subregional differences in GABAA receptor subunit expression in the rostral ventrolateral medulla of sedentary versus physically active rats.

Neurons in the rostral ventrolateral medulla (RVLM) regulate blood pressure through direct projections to spinal sympathetic preganglionic neurons. Only some RVLM neurons are active under resting conditions due to significant, tonic inhibition by gamma-aminobutyric acid (GABA). Withdrawal of GABAA receptor-mediated inhibition of the RVLM increases sympathetic outflow and blood pressure substantially, providing a mechanism by which the RVLM could contribute chronically to cardiovascular disease (CVD). Here, we tested the hypothesis that sedentary conditions, a major risk factor for CVD, increase GABAA receptors in RVLM, including its rostral extension (RVLMRE ), both of which contain bulbospinal catecholamine (C1) and non-C1 neurons. We examined GABAA receptor subunits GABAAα1 and GABAAα2 in the RVLM/RVLMRE of sedentary or physically active (10-12 weeks of wheel running) rats. Western blot analyses indicated that sedentary rats had lower expression of GABAAα1 and GABAAα2 subunits in RVLM but only GABAAα2 was lower in the RVLMRE of sedentary rats. Sedentary rats had significantly reduced expression of the chloride transporter, KCC2, suggesting less effective GABA-mediated inhibition compared to active rats. Retrograde tracing plus triple-label immunofluorescence identified fewer bulbospinal non-C1 neurons immunoreactive for GABAAα1 but a higher percentage of bulbospinal C1 neurons immunoreactive for GABAAα1 in sedentary animals. Sedentary conditions did not significantly affect the number of bulbospinal C1 or non-C1 neurons immunoreactive for GABAAα2 . These results suggest a complex interplay between GABAA receptor expression by spinally projecting C1 and non-C1 neurons and sedentary versus physically active conditions. They also provide plausible mechanisms for both enhanced sympathoexcitatory and sympathoinhibitory responses following sedentary conditions.

Development of manganese-enhanced magnetic resonance imaging of the rostral ventrolateral medulla of conscious rats: Importance of normalization and comparison with other regions of interest.

Spinally projecting neurons in the rostral ventrolateral medulla (RVLM) are believed to contribute to pathophysiological alterations in sympathetic nerve activity and the development of cardiovascular disease. The ability to identify changes in the activity of RVLM neurons in conscious animals and humans, especially longitudinally, would represent a clinically important advancement in our understanding of the contribution of the RVLM to cardiovascular disease. To this end, we describe the initial development of manganese-enhanced magnetic resonance imaging (MEMRI) for the rat RVLM. Manganese (Mn2+ ) has been used to estimate in vivo neuronal activity in other brain regions because of both its paramagnetic properties and its entry into and accumulation in active neurons. In this initial study, our three goals were as follows: (1) to validate that Mn2+ enhancement occurs in functionally and anatomically localized images of the rat RVLM; (2) to quantify the dose and time course dependence of Mn2+ enhancement in the RVLM after one systemic injection in conscious rats (66 or 33 mg/kg, intraperitoneally); and (3) to compare Mn2+ enhancement in the RVLM with other regions to determine an appropriate method of normalization of T1 -weighted images. In our proof-of-concept and proof-of-principle studies, Mn2+ was identified by MRI in the rat RVLM after direct microinjection or via retrograde transport following spinal cord injections, respectively. Systemic injections in conscious rats produced significant Mn2+ enhancement at 24 h (p < 0.05). Injections of 66 mg/kg produced greater enhancement than 33 mg/kg in the RVLM and paraventricular nucleus of the hypothalamus (p < 0.05 for both), but only when normalized to baseline scans without Mn2+ injection. Consistent with findings from our previous functional and anatomical studies demonstrating subregional neuroplasticity, Mn2+ enhancement was higher in the rostral regions of the RVLM (p < 0.05). Together with important technical considerations, our studies support the development of MEMRI as a potential method to examine RVLM activity over time in conscious animal subjects.

The link between exercise and titin passive stiffness.

NEW FINDINGS: What is the topic of this review? This review focuses on how in vivo and molecular measurements of cardiac passive stiffness can predict exercise tolerance and how exercise training can reduce cardiac passive stiffness. What advances does it highlight? This review highlights advances in understanding the relationship between molecular (titin-based) and in vivo (left ventricular) passive stiffness, how passive stiffness modifies exercise tolerance, and how exercise training may be therapeutic for cardiac diseases with increased passive stiffness. Exercise can help alleviate the negative effects of cardiovascular disease and cardiovascular co-morbidities associated with sedentary behaviour; this may be especially true in diseases that are associated with increased left ventricular passive stiffness. In this review, we discuss the inverse relationship between exercise tolerance and cardiac passive stiffness. Passive stiffness is the physical property of cardiac muscle to produce a resistive force when stretched, which, in vivo, is measured using the left ventricular end diastolic pressure-volume relationship or is estimated using echocardiography. The giant elastic protein titin is the major contributor to passive stiffness at physiological muscle (sarcomere) lengths. Passive stiffness can be modified by altering titin isoform size or by post-translational modifications. In both human and animal models, increased left ventricular passive stiffness is associated with reduced exercise tolerance due to impaired diastolic filling, suggesting that increased passive stiffness predicts reduced exercise tolerance. At the same time, exercise training itself may induce both short- and long-term changes in titin-based passive stiffness, suggesting that exercise may be a treatment for diseases associated with increased passive stiffness. Direct modification of passive stiffness to improve exercise tolerance is a potential therapeutic approach. Titin passive stiffness itself may be a treatment target based on the recent discovery of RNA binding motif 20, which modifies titin isoform size and passive stiffness. Translating these discoveries that link exercise and left ventricular passive stiffness may provide new methods to enhance exercise tolerance and treat patients with cardiovascular disease.

Sedentary conditions and enhanced responses to GABA in the RVLM: role of the contralateral RVLM.

A sedentary lifestyle is a major risk factor for cardiovascular disease, and both conditions are associated with overactivity of the sympathetic nervous system. Ongoing discharge of sympathetic nerves is regulated by the rostral ventrolateral medulla (RVLM), which in turn is modulated by the primary excitatory and inhibitory neurotransmitters glutamate and γ-amino-butyric acid (GABA), respectively. We reported previously that sedentary conditions enhance GABAergic modulation of sympathoexcitation in the RVLM, despite overall increased sympathoexcitation. Thus the purpose of this study was to test the hypothesis that sedentary conditions increase responsiveness to GABA in RVLM. Male Sprague-Dawley rats performed either chronic wheeling running or remained sedentary for 12-15 wk. Animals were instrumented to perform RVLM microinjections under Inactin anesthesia while mean arterial pressure (MAP) and splanchnic sympathetic nerve activity (SSNA) were recorded. Unilateral microinjections of GABA (30 nl, 0.3-600 mM) into the RVLM produced dose-dependent decreases in MAP and SSNA; however, no group differences were observed. Inhibition of the contralateral RVLM (muscimol, 2 mM, 90 nl) caused decreases in MAP and SSNA that were not different between groups but enhanced decreases in SSNA to GABA in sedentary rats only. In sinoaortic denervated rats, GABA microinjections before or after inhibition of the contralateral RVLM caused decreases in MAP and SSNA that were not different between groups. Our results suggest that the contralateral RVLM plays an important role in buffering responses to inhibition of the ipsilateral RVLM under sedentary but not physically active conditions. Based on these studies and others, sedentary conditions appear to enhance both sympathoinhibitory and sympathoexcitatory mechanisms in the RVLM. Enhanced sympathoinhibition may act to reduce already elevated sympathetic nervous system activity following sedentary conditions.

Integration of Central and Peripheral Regulation of the Circulation during Exercise: Acute and Chronic Adaptations.

Physical movement lasting any more than a few seconds (e.g., exercise), requires coordination of motor control with concomitant changes in the cardiovascular and respiratory support necessary to respond to the rapid increases in metabolic demand. Without such coordination, delivery of oxygen and removal of waste products become rate limiting and will restrict the duration, speed, and quality of movement. Fortunately, under healthy conditions, the central and peripheral nervous systems contribute importantly to this remarkable level of coordination via complex mechanisms that remain to be fully elucidated. The purposes of this review are to present the current state of knowledge regarding: (i) mechanisms by which the body maintains appropriate perfusion pressure to all organs during acute bouts of exercise, and (ii) alterations occurring in these mechanisms via central nervous system adaptations when exercise is performed or not performed on a regular basis (e.g., physically active versus sedentary lifestyle, respectively). Results from studies performed in humans and laboratory animals provide the reader a well-rounded knowledge base. They are intended to instill an appreciation of what is known, and not known, about how the brain regulates the cardiovascular system during acute bouts of exercise, and the adaptations that occur when individuals exercise regularly versus when chronically sedentary. Discussion of the latter is intended to provide novel mechanisms for the increased incidence of cardiovascular disease in sedentary individuals versus a reduced incidence in individuals who are regularly active. © 2018 American Physiological Society. Compr Physiol 8:103-151, 2018.

Altered Differential Control of Sympathetic Outflow Following Sedentary Conditions: Role of Subregional Neuroplasticity in the RVLM.

Despite the classically held belief of an "all-or-none" activation of the sympathetic nervous system, differential responses in sympathetic nerve activity (SNA) can occur acutely at varying magnitudes and in opposing directions. Sympathetic nerves also appear to contribute differentially to various disease states including hypertension and heart failure. Previously we have reported that sedentary conditions enhanced responses of splanchnic SNA (SSNA) but not lumbar SNA (LSNA) to activation of the rostral ventrolateral medulla (RVLM) in rats. Bulbospinal RVLM neurons from sedentary rats also exhibit increased dendritic branching in rostral regions of the RVLM. We hypothesized that regionally specific structural neuroplasticity would manifest as enhanced SSNA but not LSNA following activation of the rostral RVLM. To test this hypothesis, groups of physically active (10-12 weeks on running wheels) or sedentary, male Sprague-Dawley rats were instrumented to record mean arterial pressure, LSNA and SSNA under Inactin anesthesia and during microinjections of glutamate (30 nl, 10 mM) into multiple sites within the RVLM. Sedentary conditions enhanced SSNA but not LSNA responses and SSNA responses were enhanced at more central and rostral sites. Results suggest that enhanced SSNA responses in rostral RVLM coincide with enhanced dendritic branching in rostral RVLM observed previously. Identifying structural and functional neuroplasticity in specific populations of RVLM neurons may help identify new treatments for cardiovascular diseases, known to be more prevalent in sedentary individuals.

(In)activity-related neuroplasticity in brainstem control of sympathetic outflow: unraveling underlying molecular, cellular, and anatomical mechanisms.

More people die as a result of physical inactivity than any other preventable risk factor including smoking, high cholesterol, and obesity. Cardiovascular disease, the number one cause of death in the United States, tops the list of inactivity-related diseases. Nevertheless, the vast majority of Americans continue to make lifestyle choices that are creating a rapidly growing burden of epidemic size and impact on the United States healthcare system. It is imperative that we improve our understanding of the mechanisms by which physical inactivity increases the incidence of cardiovascular disease and how exercise can prevent or rescue the inactivity phenotype. The current review summarizes research on changes in the brain that contribute to inactivity-related cardiovascular disease. Specifically, we focus on changes in the rostral ventrolateral medulla (RVLM), a critical brain region for basal and reflex control of sympathetic activity. The RVLM is implicated in elevated sympathetic outflow associated with several cardiovascular diseases including hypertension and heart failure. We hypothesize that changes in the RVLM contribute to chronic cardiovascular disease related to physical inactivity. Data obtained from our translational rodent models of chronic, voluntary exercise and inactivity suggest that functional, anatomical, and molecular neuroplasticity enhances glutamatergic neurotransmission in the RVLM of sedentary animals. Collectively, the evidence presented here suggests that changes in the RVLM resulting from sedentary conditions are deleterious and contribute to cardiovascular diseases that have an increased prevalence in sedentary individuals. The mechanisms by which these changes occur over time and their impact are important areas for future study.

Physical activity correlates with glutamate receptor gene expression in spinally-projecting RVLM neurons: a laser capture microdissection study.

Physical inactivity is an important risk factor in the development of cardiovascular disease. The rostral ventrolateral portion of the medulla (RVLM) is composed of heterogeneous populations of neurons that are involved in the regulation of the cardiovascular system. Because of functional heterogeneity, studying the changes in the gene expression of this specific population of neurons within the RVLM is challenging. In the present study, a fluorescent retrograde tracer was injected into the spinal cord to specifically label bulbospinal RVLM neurons in sedentary and active rats. Laser capture microdissection (LCM) was then employed to collect the fluorescently labeled neurons from sections encompassing the rostrocaudal extent of the RVLM. RNA extracted from the neurons was used in qRT-PCR analysis. Changes in gene expression levels of glutamate and GABA receptor subunits were compared between sedentary and physically active rats. GLUR3 subunit showed a significant negative correlation between total running distance and its relative gene expression in active rats. There were no significant difference in the gene expression of NMDA (NR1, NR2A, NR2B, NR2C and NR2D), AMPA (GLUR1, GLUR2 and GLUR3) and GABAA (GABAA1 and GABAA2) receptor subunits. Overall, the present study demonstrates the feasibility of utilizing LCM to investigate the gene expression changes in a specific population of neurons in the RVLM. Correlation studies suggest that physical activity could contribute to neuroplasticity in the RVLM.

Physical (in)activity-dependent structural plasticity in bulbospinal catecholaminergic neurons of rat rostral ventrolateral medulla.

Increased activity of the sympathetic nervous system is thought to play a role in the development and progression of cardiovascular disease. Recent work has shown that physical inactivity versus activity alters neuronal structure in brain regions associated with cardiovascular regulation. Our physiological studies suggest that neurons in the rostral ventrolateral medulla (RVLM) are more responsive to excitation in sedentary versus physically active animals. We hypothesized that enhanced functional responses in the RVLM may be due, in part, to changes in the structure of RVLM neurons that control sympathetic activity. We used retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) to identify bulbospinal catecholaminergic (C1) neurons in sedentary and active rats after chronic voluntary wheel-running exercise. We then digitally reconstructed their cell bodies and dendrites at different rostrocaudal levels. The dendritic arbors of spinally projecting TH neurons from sedentary rats were more branched than those of physically active rats (P < 0.05). In sedentary rats, dendritic branching was greater in more rostral versus more caudal bulbospinal C1 neurons, whereas, in physically active rats, dendritic branching was consistent throughout the RVLM. In contrast, cell body size and the number of primary dendrites did not differ between active and inactive animals. We suggest that these structural changes provide an anatomical underpinning for the functional differences observed in our in vivo studies. These inactivity-related structural and functional changes may enhance the overall sensitivity of RVLM neurons to excitatory stimuli and contribute to an increased risk of cardiovascular disease in sedentary individuals.

Immunoreactivity for the NMDA NR1 subunit in bulbospinal catecholamine and serotonin neurons of rat ventral medulla.

Bulbospinal neurons in the ventral medulla play important roles in the regulation of sympathetic outflow. Physiological evidence suggests that these neurons are activated by N-methyl-D-aspartate (NMDA) and non-NMDA subtypes of glutamate receptors. In this study, we examined bulbospinal neurons in the ventral medulla for the presence of immunoreactivity for the NMDA NR1 subunit, which is essential for NMDA receptor function. Rats received bilateral injections of cholera toxin B into the tenth thoracic spinal segment to label bulbospinal neurons. Triple immunofluorescent labeling was used to detect cholera toxin B with a blue fluorophore, NR1 with a red fluorophore, and either tyrosine hydroxylase or tryptophan hydroxylase with a green fluorophore. In the rostral ventrolateral medulla, NR1 occurred in all bulbospinal tyrosine hydroxylase-positive neurons and 96% of bulbospinal tyrosine hydroxylase-negative neurons, which were more common in sections containing the facial nucleus. In the raphe pallidus, the parapyramidal region, and the marginal layer, 98% of bulbospinal tryptophan hydroxylase-positive neurons contained NR1 immunoreactivity. NR1 was also present in all of the bulbospinal tryptophan hydroxylase-negative neurons, which comprised 20% of bulbospinal neurons in raphe pallidus and the parapyramidal region. These results show that virtually all bulbospinal tyrosine hydroxylase and non-tyrosine hydroxylase neurons in the rostral ventrolateral medulla and virtually all bulbospinal serotonin and non-serotonin neurons in raphe pallidus and the parapyramidal region express NR1, the obligatory subunit of the NMDA receptor. NMDA receptors on bulbospinal neurons in the rostral ventral medulla likely influence sympathoexcitation in normal and pathological conditions.

Selective enhancement of glutamate-mediated pressor responses after GABA(A) receptor blockade in the RVLM of sedentary versus spontaneous wheel running rats.

Overactivity of the sympathetic nervous system (SNS) is a hallmark of many cardiovascular diseases. It is also well-known that physical inactivity independently contributes to cardiovascular diseases, likely in part via increased SNS activity. Recent work from our laboratory has demonstrated increased SNS responses in sedentary animals following either direct activation or disinhibition of the rostral ventrolateral medulla (RVLM), an integral cardiovascular brainstem region. These data led us to hypothesize that the interaction between excitation and inhibition of the RVLM is altered in sedentary versus physically active animals. To test this hypothesis, we recorded mean arterial pressure (MAP) and lumbar sympathetic nerve activity (LSNA) in Inactin anesthetized rats that were housed for 8-12 weeks with or without access to a running wheel. Pressor responses to direct activation of the RVLM with glutamate were similar between groups under intact conditions. However, blockade of γ-aminobutyric acid (GABA)(A) receptors with bicuculline selectively enhanced pressor responses to glutamate in sedentary animals. Interestingly, LSNA responses to glutamate were not enhanced in sedentary versus active animals in the presence or absence of tonic GABAergic tone. These results suggest that sedentary compared to active conditions enhance GABAergic inhibition of glutamate-sensitive neurons in the RVLM that are involved in blood pressure regulation, and by mechanisms that do not involve LSNA. We also speculate that regular physical activity has differential effects on SNS activity to specific vascular beds and may reduce the risk of developing cardiovascular diseases via changes occurring in the RVLM.

Regulation of arterial pressure by the paraventricular nucleus in conscious rats: interactions among glutamate, GABA, and nitric oxide.

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation. Experiments in anesthetized animals and in vitro indicate an interaction among gamma-aminobutyric acid (GABA), nitric oxide (NO), and glutamate in the PVN. The cardiovascular role of the PVN and interactions of these neurotransmitters in conscious animals have not been evaluated fully. In chronically instrumented conscious rats, mean arterial pressure (MAP) and heart rate (HR) responses to microinjections (100 nl) in the region of the PVN were tested. Bilateral blockade of ionotropic excitatory amino acid (EAA) receptors (kynurenic acid, Kyn) in the PVN produced small but significant decreases in MAP and HR. GABA(A) receptor blockade (bicuculline, Bic), and inhibition of NO synthase [(NOS), N-(G)-monomethyl-L-arginine, L-NMMA] each increased MAP and HR. The NO donor sodium nitroprusside (SNP) produced depressor responses that were attenuated by Bic. NOS inhibition potentiated both pressor responses to the selective EAA agonist, N-methyl-D-aspartic acid (NMDA), and depressor responses to Kyn. Increases in MAP and HR due to Bic were blunted by prior blockade of EAA receptors. Thus, pressor responses to GABA blockade require EAA receptors and GABA neurotransmission contributes to NO inhibition. Tonic excitatory effects of glutamate in the PVN are tonically attenuated by NO. These data demonstrate that, in the PVN of conscious rats, GABA, glutamate, and NO interact in a complex fashion to regulate arterial pressure and HR under normal conditions.