Hypothalamic Orexinergic Neurons Projecting to the Mesencephalic Locomotor Region Are Activated by Voluntary Wheel Running Exercise in Rats.

Background: Cardiovascular changes during exercise are regulated by a motor volitional signal, called central command, which originates in the rostral portions of the brain and simultaneously regulates somatomotor and autonomic nervous systems. Whereas we recently elucidated mesencephalic locomotor region (MLR) neurons projecting to the rostral ventrolateral medulla as a crucial component of the central circuit responsible for transmitting central command signals, upstream circuits that regulate the MLR neurons remain unknown. Orexinergic neurons, which primarily originate from the perifornical area (PeFA) of the hypothalamus and reportedly play roles in eliciting locomotion and elevating sympathetic activity, send axonal projection to the MLR. The knowledge led us to investigate whether central command signals are relayed through orexinergic neurons projecting to the MLR. Methods: We performed anterograde transsynaptic tagging with AAV1 encoding Cre to confirm the presence of MLR neurons postsynaptic to the PeFA in rats. We also conducted retrograde neural tracing with retrograde AAV, combined with immunohistochemical staining, to examine the excitability of MLR-projecting orexinergic neurons in rats that were allowed to freely run on the wheel for 90 min. Results: A significant number of MLR neurons were labeled with Cre, indicating that PeFA neurons make synaptic contacts with MLR neurons. Moreover, immunoreactivities of Fos, a marker of neuronal excitation, were found in many MLR-projecting orexinergic neurons by voluntary wheel running exercise, compared to non-exercising control rats, especially in the intermediate-posterior, rather than anterior, and medial, rather than lateral, portions within the orexinergic neuron-distributing domain. Conclusion: The findings suggest that specifically located orexinergic neurons transmit central command signals onto the MLR for running exercise. Elucidating the role of these MLR-projecting orexinergic neurons in somatomotor control and autonomic cardiovascular control deserves further study to unveil central circuit mechanisms responsible for central command function.

Generation of c-Fos knockout rats, and observation of their phenotype.

c-Fos is a useful marker gene of neuron activation for neuroscience and physiology research. The mechanism and function of neural networks have been elucidated using c-Fos reporter knock-in (KI) mice, but the small size of the mice makes it difficult to perform surgical procedures on specific brain regions. On the other hand, there is a large amount of accumulated data on behavioral studies using rats. Thus, the generation of c-Fos reporter rat is expected, but it is difficult to generate gene-modified rats. Furthermore, c-Fos gene abnormality is expected to be severe in rats, as shown in homozygous of c-Fos knockout (KO) mouse, but such analysis has rarely been performed and is not certain. This study generated c-Fos-deficient rats using CRISPR/Cas, with 1067 bp deletion including exon 1 of the c-Fos gene. Homozygous c-Fos KO rats had growth latency and the same tooth and bone abnormality as homozygous c-Fos KO mice but not heterozygous c-Fos KO rats. Therefore, the c-Fos gene in rats is expected to have the same function as that in mice, and the generation of c-Fos reporter KI rats is further anticipated.

A brainstem monosynaptic excitatory pathway that drives locomotor activities and sympathetic cardiovascular responses.

Exercise including locomotion requires appropriate autonomic cardiovascular adjustments to meet the metabolic demands of contracting muscles, yet the functional brain architecture underlying these adjustments remains unknown. Here, we demonstrate brainstem circuitry that plays an essential role in relaying volitional motor signals, i.e., central command, to drive locomotor activities and sympathetic cardiovascular responses. Mesencephalic locomotor neurons in rats transmit central command-driven excitatory signals onto the rostral ventrolateral medulla at least partially via glutamatergic processes, to activate both somatomotor and sympathetic nervous systems. Optogenetic excitation of this monosynaptic pathway elicits locomotor and cardiovascular responses as seen during running exercise, whereas pathway inhibition suppresses the locomotor activities and blood pressure elevation during voluntary running without affecting basal cardiovascular homeostasis. These results demonstrate an important subcortical pathway that transmits central command signals, providing a key insight into the central circuit mechanism required for the physiological conditioning essential to maximize exercise performance.

α1-Adrenergic receptor mediates adipose-derived stem cell sheet-induced protection against chronic heart failure after myocardial infarction in rats.

Cell-based therapy using adipose-derived stem cells (ADSCs) has emerged as a novel therapeutic approach to treat heart failure after myocardial infarction (MI). The purpose of this study was to determine whether inhibition of α1-adrenergic receptors (α1-ARs) in ADSCs attenuates ADSC sheet-induced improvements in cardiac functions and inhibition of remodeling after MI. ADSCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ADSCs, we determined the mRNA levels of vascular endothelial growth factor (VEGF)-A and α1-AR under normoxia or hypoxia and the effects of norepinephrine and an α1-blocker, doxazosin, on the mRNA levels of angiogenic factors. Hypoxia increased α1-AR and VEGF mRNA levels in ADSCs. Norepinephrine further increased VEGF mRNA expression under hypoxia; this effect was abolished by doxazosin. Tube formation of human umbilical vein endothelial cells was promoted by conditioned media of ADSCs treated with the α1 stimulant phenylephrine under hypoxia but not by those of ADSCs pretreated with phenylephrine plus doxazosin. In in vivo studies using rats with MI, transplanted ADSC sheets improved cardiac functions, facilitated neovascularization, and suppressed fibrosis after MI. These effects were abolished by doxazosin treatment. Pathway analysis from RNA sequencing data predicted significant upregulation of α1-AR mRNA expression in transplanted ADSC sheets and the involvement of α1-ARs in angiogenesis through VEGF. In conclusion, doxazosin abolished the beneficial effects of ADSC sheets on rat MI hearts as well as the enhancing effect of norepinephrine on VEGF expression in ADSCs, indicating that ADSC sheets promote angiogenesis and prevent cardiac dysfunction and remodeling after MI via their α1-ARs.

Sympathoexcitatory input from hypothalamic paraventricular nucleus neurons projecting to rostral ventrolateral medulla is enhanced after myocardial infarction.

Elevated sympathetic vasomotor tone seen in heart failure (HF) may involve dysfunction of the hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). This study aimed to elucidate the role of PVN-RVLM neurons in the maintenance of resting renal sympathetic nerve activity (RSNA) after myocardial infarction (MI). In male rats, the left coronary artery was chronically ligated to induce MI. The rats received PVN microinjections of an adeno-associated viral (AAV) vector encoding archaerhodopsin T (ArchT) with the reporter yellow fluorescence protein (eYFP). The ArchT rats had abundant distributions of eYFP-labeled, PVN-derived axons in the RVLM. In anesthetized ArchT rats with MI (n = 12), optogenetic inhibition of the PVN-RVLM pathway achieved by 532-nm-wavelength laser illumination to the RVLM significantly decreased RSNA. This effect was not found in sham-operated ArchT rats (n = 6). Other rat groups received RVLM microinjections of a retrograde AAV vector encoding the red light-drivable halorhodopsin Jaws (Jaws) with the reporter green fluorescence protein (GFP) and showed expression of GFP-labeled cell bodies and dendrites in the PVN. Laser illumination of the PVN at a 635 nm wavelength elicited significant renal sympathoinhibition in Jaws rats with MI (n = 9) but not in sham-operated Jaws rats (n = 8). These results indicate that sympathoexcitatory input from PVN-RVLM neurons is enhanced after MI, suggesting that this monosynaptic pathway is part of the central nervous system circuitry that plays a critical role in generating an elevated sympathetic vasomotor tone commonly seen with HF.NEW & NOTEWORTHY Using optogenetics in rats, we report that sympathoexcitatory input from hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla is enhanced after myocardial infarction. It is suggested that this monosynaptic pathway makes up a key part of central nervous system circuitry underlying sympathetic hyperactivation commonly seen in heart failure.

Augmented fear bradycardia in rats with heart failure.

In congestive heart failure (CHF), while resting parasympathetic activity becomes reduced, parasympathetically-mediated responses to stressors have not been described. This study aimed to (1) elucidate the effect of CHF on fear bradycardia, a parasympathetically-mediated response, and (2) examine if brain oxidative stress of CHF mediates fear bradycardia. White noise sound (WNS) exposure to conscious rats induced freezing behavior and elicited bradycardia. WNS exposure-elicited bradycardia was greater in rats with CHF than in controls. Superoxide dismutase mimetics administered in the lateral/ventrolateral midbrain periaqueductal gray (l/vlPAG), a region that contributes to the generation of fear bradycardia, had no effect on the bradycardia response in control and CHF rats. Dihydroethidium staining in situ showed that superoxide generation in the l/vlPAG of CHF rats was increased as compared to controls. These results demonstrate that CHF leads to the augmentation of fear bradycardia. Moreover, oxidative stress in the l/vlPAG of CHF unlikely mediates the augmented fear bradycardia.

Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

KEY POINTS: Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN-RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. ABSTRACT: The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN-RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN-RVLM neurons. Male rats received microinjections into the PVN with an adeno-associated virus (AAV) vector that encoded a hybrid of channelrhodopsin-2/1 with the reporter tdTomato (ChIEF-tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2-GFPretro ). Under anaesthesia with urethane and α-chloralose, photostimulation (473 nm wavelength) of PVN-RVLM neurons, achieved by laser illumination of either RVLM of ChIEF-tdTomato rats (n = 8) or PVN of ChR2-GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF-tdTomato rats were closely associated with tdTomato-labelled, PVN-derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF-tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN-RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons.

Angiotensin II, Oxidative Stress, and Sympathetic Nervous System Hyperactivity in Heart Failure.

In congestive heart failure (CHF), sympathetic nervous system is hyperactive. This article reviews current understandings about central and peripheral neural mechanisms underlying sympathetic hyperactivation in this pathological condition. During the development of CHF, renin-angiotensin system (RAS) activities and angiotensin II-mediated oxidative stress become enhanced. Here, on the basis of findings obtained from animal studies, it is examined how RAS overactivation and oxidative stress in central and peripheral nervous systems of CHF mediate sympathetic hyperactivation. Mechanisms by which exercise training in CHF ameliorates RAS overactivation, oxidative stress and sympathetic hyperactivation are also investigated.

Distribution of Fos-immunoreactive cells in the ventral part of rat medulla following voluntary treadmill exercise.

The ventral part of the medulla, which contains important cardiovascular regions, is reportedly activated during exercise. Nevertheless, it was uncertain which region(s) in the ventral medulla are specifically activated by exercise. The present study aimed to demonstrate a general pattern of exercise-specific distribution of excited neuronal cells in the rat ventral medulla. Via immunohistochemical experiments, we mapped tyrosine hydroxylase- and Fos-immunoreactive cells (TH-IR and Fos-IR cells, respectively) on rat medullary coronal sections following a bout of voluntary treadmill exercise, a comparative control period, or after pharmacologically induced-hypotension under anesthesia. In the ventral medulla at the rostrocaudal level adjacent, but not rostral or caudal, to the caudal edge of the facial nucleus, voluntary treadmill exercise induced significant (P<0.05) increases in Fos expression, similar to hypotension. The rostral ventrolateral medulla (RVLM), as compared with the rostral ventromedial medulla (RVMM), displayed a greater number of Fos-IR cells due to either exercise or hypotension. In the RVLM, either exercise or hypotension induced significant expression of Fos in both TH-IR and TH non-immunoreactive cells. In the caudal ventrolateral medulla (CVLM), hypotension, but not exercise, increased the ratio of Fos-IR cells in the TH-IR population. These findings demonstrate that RVLM adrenergic and non-adrenergic neurons are specifically excited by voluntary exercise in rats, while RVMM or CVLM neurons are not. We suggest that RVLM C1/non-C1 neurons are a major part of central circuitries underlying sympathetic adjustments to exercise.

Role played by periaqueductal gray neurons in parasympathetically mediated fear bradycardia in conscious rats.

Freezing, a characteristic pattern of defensive behavior elicited by fear, is associated with a decrease in the heart rate. Central mechanisms underlying fear bradycardia are poorly understood. The periaqueductal gray (PAG) in the midbrain is known to contribute to autonomic cardiovascular adjustments associated with various emotional behaviors observed during active or passive defense reactions. The purpose of this study was to elucidate the role played by PAG neurons in eliciting fear bradycardia. White noise sound (WNS) exposure at 90 dB induced freezing behavior and elicited bradycardia in conscious rats. The WNS exposure-elicited bradycardia was mediated parasympathetically because intravenous administration of atropine abolished the bradycardia (P < 0.05). Moreover, WNS exposure-elicited bradycardia was mediated by neuronal activation of the lateral/ventrolateral PAG (l/vlPAG) because bilateral microinjection of muscimol, a GABAA agonist, into the l/vlPAG significantly suppressed the bradycardia. It is noted that muscimol microinjected bilaterally into the dorsolateral PAG had no effect on WNS exposure-elicited bradycardia. Furthermore, retrograde neuronal tracing experiments combined with immunohistochemistry demonstrated that a number of l/vlPAG neurons that send direct projections to the nucleus ambiguus (NA) in the medulla, a major origin of parasympathetic preganglionic neurons to the heart, were activated by WNS exposure. Based on these findings, we propose that the l/vlPAG-NA monosynaptic pathway transmits fear-driven central signals, which elicit bradycardia through parasympathetic outflow.

Central command dysfunction in rats with heart failure is mediated by brain oxidative stress and normalized by exercise training.

Sympathoexcitation elicited by central command, a parallel activation of the motor and autonomic neural circuits in the brain, has been shown to become exaggerated in chronic heart failure (CHF). The present study tested the hypotheses that oxidative stress in the medulla in CHF plays a role in exaggerating central command-elicited sympathoexcitation, and that exercise training in CHF suppresses central command-elicited sympathoexcitation through its antioxidant effects in the medulla. In decerebrate rats, central command was activated by electrically stimulating the mesencephalic locomotor region (MLR) after neuromuscular blockade. The MLR stimulation at a current intensity greater than locomotion threshold in rats with CHF after myocardial infarction (MI) evoked larger (P < 0.05) increases in renal sympathetic nerve activity and arterial pressure than in sham-operated healthy rats (Sham) and rats with CHF that had completed longterm (8­12 weeks) exercise training (MI + TR). In the Sham and MI + TR rats, bilateral microinjection of a superoxide dismutase (SOD) mimetic Tempol into the rostral ventrolateral medulla (RVLM) had no effects on MLR stimulation-elicited responses. By contrast, in MI rats, Tempol treatment significantly reduced MLR stimulation-elicited responses. In a subset of MI rats, treatment with Tiron, another SOD mimetic, within the RVLM also reduced responses. Superoxide generation in the RVLM, as evaluated by dihydroethidium staining, was enhanced in MI rats compared with that in Sham and MI + TR rats. Collectively, these results support the study hypotheses. We suggest that oxidative stress in the medulla in CHF mediates central command dysfunction, and that exercise training in CHF is capable of normalizing central command dysfunction through its antioxidant effects in the medulla.

Oxidative stress exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation in rats with hypertension induced by angiotensin II.

Muscle contraction stimulates thin fiber muscle afferents and evokes reflex sympathoexcitation. In hypertension, this reflex is exaggerated. ANG II, which is elevated in hypertension, has been reported to trigger the production of superoxide and other reactive oxygen species. In the present study, we tested the hypothesis that increased ANG II in hypertension exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation by inducing oxidative stress in the muscle. In rats, subcutaneous infusion of ANG II at 450 ng·kg(-1)·min(-1) for 14 days significantly (P < 0.05) elevated blood pressure compared with sham-operated (sham) rats. Electrically induced 30-s hindlimb muscle contraction in decerebrate rats with hypertension evoked larger renal sympathoexcitatory and pressor responses [+1,173 ± 212 arbitrary units (AU) and +35 ± 5 mmHg, n = 10] compared with sham normotensive rats (+419 ± 103 AU and +13 ± 2 mmHg, n = 11). Tempol, a SOD mimetic, injected intra-arterially into the hindlimb circulation significantly reduced responses in hypertensive rats, whereas this compound had no effect on responses in sham rats. Tiron, another SOD mimetic, also significantly reduced reflex renal sympathetic and pressor responses in a subset of hypertensive rats (n = 10). Generation of muscle superoxide, as evaluated by dihydroethidium staining, was increased in hypertensive rats. RT-PCR and immunoblot experiments showed that mRNA and protein for gp91(phox), a NADPH oxidase subunit, in skeletal muscle tissue were upregulated in hypertensive rats. Taken together, hese results suggest that increased ANG II in hypertension induces oxidative stress in skeletal muscle, thereby exaggerating the muscle reflex.

Exercise pressor reflex function in female rats fluctuates with the estrous cycle.

In women, sympathoexcitation during static handgrip exercise is reduced during the follicular phase of the ovarian cycle compared with the menstrual phase. Previous animal studies have demonstrated that estrogen modulates the exercise pressor reflex, a sympathoexcitatory mechanism originating in contracting skeletal muscle. The present study was conducted in female rats to determine whether skeletal muscle contraction-evoked reflex sympathoexcitation fluctuates with the estrous cycle. The estrous cycle was judged by vaginal smear. Plasma concentrations of estrogen were significantly (P < 0.05) higher in rats during the proestrus phase of the estrus cycle than those during the diestrus phase. In decerebrate rats, either electrically induced 30-s continuous static contraction of the hindlimb muscle or 30-s passive stretch of Achilles tendon (a maneuver that selectively stimulates mechanically sensitive muscle afferents) evoked less renal sympathoexcitatory and pressor responses in the proestrus animals than in the diestrus animals. Renal sympathoexcitatory response to 1-min intermittent (1- to 4-s stimulation to relaxation) bouts of static contraction was also significantly less in the proestrus rats than that in the diestrus rats. In ovariectomized female rats, 17ß-estradiol applied into a well covering the dorsal surface of the lumbar spinal cord significantly reduced skeletal muscle contraction-evoked responses. These observations demonstrate that the exercise pressor reflex function and its mechanical component fluctuate with the estrous cycle in rats. Estrogen may cause these fluctuations through its attenuating effects on the spinal component of the reflex arc.

Green odor and depressive-like state in rats: toward an evidence-based alternative medicine?

It is widely accepted that mental stress is an important factor in the development of psychological disorders such as depression. On pre-existing evidence, the so-called green odor may have a relieving and sedative effect on animals exposed to stressful situations. Using two behavioral models of depression, the forced-swim test and learned helplessness paradigm, we investigated whether inhalation of green odor (a 50:50 mixture of trans-2-hexenal and cis-3-hexenol) might alleviate and/or prevent experimentally induced depressive-like states in rats. A 3-min swim every day for 7 days resulted in significant prolongation of immobility time (vs. day 1). Inhaling green odor, but not vehicle, thereafter for 10 days (without swimming) led to the prolonged immobility time being significantly reduced and the hippocampal level of brain-derived neurotrophic factor (BDNF) being significantly increased. In the learned helplessness paradigm, the failure number and time spent in the shock compartment seen in the active avoidance test were both significantly attenuated in those rats that inhaled green odor for 11 days after the postshock screening test (vs. vehicle-exposed rats). Finally, for 10 consecutive days rats continuously exposed to green odor or vehicle swam for 3 min/day. Immobility time was significantly shorter in the green-odor group than in the vehicle-exposed group on days 6-10. These results suggest that green odor has not only a therapeutic, but also a preventive effect on depressive-like states in rats. These effects may be at least in part due to a green odor-induced upregulation of BDNF in the hippocampus.

Transient receptor potential A1 channel contributes to activation of the muscle reflex.

This study was undertaken to elucidate the role played by transient receptor potential A1 channels (TRPA1) in activating the muscle reflex, a sympathoexcitatory drive originating in contracting muscle. First, we tested the hypothesis that stimulation of the TRPA1 located on muscle afferents reflexly increases sympathetic nerve activity. In decerebrate rats, allyl isothiocyanate, a TRPA1 agonist, was injected intra-arterially into the hindlimb muscle circulation. This led to a 33% increase in renal sympathetic nerve activity (RSNA). The effect of allyl isothiocyanate was a reflex because the response was prevented by sectioning the sciatic nerve. Second, we tested the hypothesis that blockade of TRPA1 reduces RSNA response to contraction. Thirty-second continuous static contraction of the hindlimb muscles, induced by electrical stimulation of the peripheral cut ends of L(4) and L(5) ventral roots, increased RSNA and blood pressure. The integrated RSNA during contraction was reduced by HC-030031, a TRPA1 antagonist, injected intra-arterially (163 ± 24 vs. 95 ± 21 arbitrary units, before vs. after HC-030031, P < 0.05). Third, we attempted to identify potential endogenous stimulants of TRPA1, responsible for activating the muscle reflex. Increases in RSNA in response to injection into the muscle circulation of arachidonic acid, bradykinin, and diprotonated phosphate, which are metabolic by-products of contraction and stimulants of muscle afferents during contraction, were reduced by HC-030031. These observations suggest that the TRPA1 located on muscle afferents is part of the muscle reflex and further support the notion that arachidonic acid metabolites, bradykinin, and diprotonated phosphate are candidates for endogenous agonists of TRPA1.

Chronic femoral artery occlusion augments exercise pressor reflex in decerebrated rats.

In decerebrated rats, we determined the pressor and cardioaccelerator reflex responses to static contraction of hindlimb muscles whose femoral arteries were either occluded 72 h before contraction, occluded 3 min before contraction, or freely perfused. We found that the pressor reflex arising from the limb whose femoral artery was occluded for 72 h before contraction (32 +/- 5 mmHg, n = 16) was significantly higher than the pressor reflex arising from the contralateral freely perfused limb (15 +/- 3 mmHg, n = 16, P < 0.001) or than that arising from the contralateral limb whose femoral artery was occluded for only 3 min (17 +/- 4 mmHg, n = 16, P < 0.001). Moreover, the pressor reflex arising from the limb whose femoral artery was occluded for 3 min before the start of contraction was not significantly different than that arising from the contralateral freely perfused limb (n = 16, P = 0.819). The pressor component of the reflex arising from the limb whose femoral artery was occluded for 72 h was not changed by transient receptor potential vanilloid (TRPV) 1 receptor blockade with iodo-resiniferatoxin (n = 15, P = 0.272), although the cardioaccelerator component was significantly reduced (P = 0.005). In addition, the pressor response evoked by capsaicin injection in the femoral artery of the 72-h occluded limb was more than double that evoked from the freely perfused limb (P = 0.026). We conclude that chronic (i.e., 72 h) but not acute (3 min), femoral arterial occlusion augments pressor reflex arising from contraction of hindlimb muscles and that TRPV1 receptors play little role in this augmentation.

Bradykinin receptor blockade reduces sympathetic nerve response to muscle contraction in rats with ischemic heart failure.

Previous animal and human studies have suggested that a muscle reflex engaged during contraction leads to heightened levels of sympathetic activity in congestive heart failure (CHF). The present experiment was designed to test the role for bradykinin, which is produced within contracting skeletal muscle and contributes to the muscle reflex through its action on kinin B(2) receptors located on the endings of thin fiber muscle afferents. CHF was induced in rats by myocardial infarction (MI) after coronary artery ligation. Echocardiography was performed to determine fractional shortening (FS), an index of the left ventricular function. In the decerebrate rats, we examined renal sympathetic nerve activity (RSNA) during 1 min intermittent (1 to 4 s stimulation to relaxation) contraction of left triceps surae muscles. RSNA responded synchronously as tension was developed, and the response was significantly (P < 0.05) greater in MI rats [+39 +/- 9% s(-1) (integrated RSNA over time); n = 16] with 20 +/- 2% of FS than that in control healthy rats (+19 +/- 2% s(-1); n = 16) with 49 +/- 2% of FS. Tension development did not differ significantly between the two groups of rats. Thirty minutes after intra-arterial injection into the hindlimb circulation of the kinin B(2) receptor antagonist, HOE-140 (2 microg/kg), the RSNA response to contraction was significantly reduced in the MI rats (+26 +/- 7% s(-1)) but not in the control rats (+17 +/- 2% s(-1)). These data suggest that bradykinin within contracting muscle is part of the exaggerated muscle reflex seen in CHF.

Oxidative stress and the muscle reflex in heart failure.

Muscle contraction stimulates thin fibre muscle afferents and evokes a reflex increase in blood pressure. In heart failure (HF) this reflex is accentuated. Of note, superoxide and other reactive oxygen species are increased in HF. In this report, we tested the hypothesis that excess superoxide contributes to the exaggerated muscle reflex in HF. HF was induced in rats by coronary artery ligation. Electrically induced 30 s hindlimb muscle contraction in decerebrate rats with myocardial infarction (MI) (left ventricular fractional shortening (FS) = 24 +/- 1%; n = 15) evoked larger (P < 0.05) increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) as compared to control rats (FS = 47 +/- 1%; n = 14). In the MI rats, the pressor and RSNA responses to contraction were reduced by intra-arterial injection into the hindlimb circulation of tempol (10 mg), a superoxide dismutase mimetic (DeltaMAP: 22 +/- 2 vs. 11 +/- 1 mmHg; integral DeltaRSNA: 1032 +/- 204 vs. 431 +/- 73 arbitrary units (a.u.); before vs. after tempol; P < 0.05). Tempol also attenuated the RSNA response to 1 min intermittent (1-4 s stimulation to relaxation) bouts of static contraction in the MI rats (116 +/- 17 vs. 72 +/- 11 a.u.; P < 0.05; n = 16). In the control rats, tempol had no effect on these responses. These results suggest that excess superoxide in HF sensitizes mechanically sensitive muscle afferents engaged during contraction. We hypothesize that oxidative stress contributes to the exaggerated muscle reflex in HF.

Spinal P2X receptor modulates muscle pressor reflex via glutamate.

Static contraction of skeletal muscle evokes reflex increases in blood pressure and heart rate. Previous studies showed that P2X receptors located at the dorsal horn of the spinal cord play a role in modulating the muscle pressor reflex. P2X stimulation can alter release of the excitatory amino acid, glutamate (Glu). In this report, we tested the hypothesis that stimulation of P2X receptors enhances the concentrations of Glu ([Glu]) in the dorsal horn, and that blocking P2X receptors attenuates contraction-induced Glu increases and the resultant reflex pressor response. Contraction was elicited by electrical stimulation of the L(7) and S(1) ventral roots of 14 cats. Glu samples were collected from microdialysis probes inserted in the L(7) level of the dorsal horn of the spinal cord, and dialysate [Glu] was determined using the HPLC method. First, microdialyzing alpha,beta-methylene ATP (0.4 mM) into the dorsal horn significantly increased [Glu]. In addition, contraction elevated [Glu] from baseline of 536 +/- 53 to 1,179 +/- 192 nM (P < 0.05 vs. baseline), and mean arterial pressure by 39 +/- 8 mmHg in the control experiment. Microdialyzing the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (10 mM) into the dorsal horn attenuated the contraction induced-Glu increase (610 +/- 128 to 759 +/- 147 nM; P > 0.05) and pressor response (16 +/- 3 mmHg, P < 0.05 vs. control). Our findings demonstrate that P2X modulates the cardiovascular responses to static muscle contraction by affecting the release of Glu in the dorsal horn of the spinal cord.

Gadolinium inhibits group III but not group IV muscle afferent responses to dynamic exercise.

Dynamic exercise has been shown to stimulate rapidly both group III and IV muscle afferents. The often rapid (i.e. 2 s) onset latencies of the group IV afferents is particularly surprising because these unmyelinated afferents are thought to respond to the gradual accumulation of metabolites signalling a mismatch between blood/oxygen demand and supply in exercising muscles. One explanation for the rapid onset to exercise by group IV afferents is that they are mechanosensitive, a concept that has been supported by the finding that these afferents were stimulated by vasodilatation induced by injection of vasoactive drugs. We therefore examined in decerebrated cats the effect of gadolinium, a blocker of mechanogated channels, on the responses of group III and IV muscle afferents to dynamic exercise induced by electrical stimulation of the mesencephalic locomotor region. We found that gadolinium (10 mm; 1 ml) injected into the abdominal aorta had no significant effect (P > 0.05) on the responses of 11 group IV afferents to dynamic exercise. In contrast, gadolinium markedly attenuated the responses of 11 group III afferents to exercise (P < 0.05). Our findings suggest that group IV afferents are not responding to a mechanical stimulus during exercise. Instead their rapid response to dynamic exercise might be caused by a chemical substance whose concentration is directly proportional to blood flow, which increases in the skeletal muscles when they are dynamically exercising.