Afferents In Rats Research Articles

Protein kinase C epsilon contributes to chronic mechanoreflex sensitization in rats with heart failure.

We investigated second-messenger signalling components linked to the stimulation of Gq protein-coupled receptors (e.g. thromboxane A2 and bradykinin B2 receptors) on the sensory endings of thin fibre muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (5µg) or the PKCε translocation inhibitor PKCe141 (45µg) into the arterial supply of the hindlimb would reduce the increase in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) evoked during 30s of 1Hz dynamic hindlimb muscle stretch in decerebrate, unanaesthetized HF-rEF rats but not sham-operated controls (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 (19)%) compared to SHAM (80 (9)%; P<0.001) rats. In HF-rEF rats (n=3M/2F), IP3 receptor blockade had no effect on the peak ΔRSNA (pre: 99 (74)%; post: 133 (79)%; P=0.974) or peak ΔMAP response to stretch (peak ΔMAP: pre: 32 (14) mmHg; post: 36 (21) mmHg; P=0.719). Conversely, in another group of HF-rEF rats (n=4M/3F), the PKCε translocation inhibitor reduced the peak ΔRSNA (pre: 110 (77)%; post: 62 (58)%; P=0.029) and peak ΔMAP response to stretch (pre: 30 (20) mmHg; post: 17 (16) mmHg; P=0.048). In SHAM counterparts, neither drug affected the mechanoreflex responses. Our findings highlight PKCε, but not IP3 receptors, as a significant second-messenger in the chronic mechanoreflex sensitization in HF-rEF which may play a crucial role in the exaggerated sympathetic response to exercise in this patient population. KEY POINTS: Skeletal muscle contraction results in an exaggerated reflex increase in sympathetic nerve activity in heart failure patients with reduced ejection fraction (HF-rEF) compared to healthy individuals, contributing to increased cardiovascular risk and impaired tolerance for mild exercise. The exaggerated reflex sympathetic responses in HF-rEF may be attributed to a chronic sensitization of mechanically sensitive thin fibre muscle afferents mediated, at least in part, by stimulation of Gq protein-coupled thromboxane A2 and bradykinin B2 receptors on muscle afferent sensory endings. The specific Gq protein-linked signalling mechanisms that produce the chronic mechanoreflex sensitization in HF-rEF have not been investigated but may involve inositol 1,4,5-trisphosphate (IP3) receptors and/or protein kinase C epsilon (PKCε). Here we demonstrate that PKCε, but not IP3 receptors, within the sensory endings of thin fibre muscle afferents plays a role in the sensitization of mechanically sensitive thin fibre muscle afferents in rats with HF-rEF.

Implications of high homocysteine levels in migraine pain: An experimental study of the excitability of peripheral meningeal afferents in rats with hyperhomocysteinemia.

Investigation of chronic homocysteine action on the excitability and N-methyl-D-aspartate (NMDA) sensitivity of the peripheral trigeminovascular system of rats. Migraine is a neurological disease that affects 15%-20% of the general population. Epidemiological observations show that an increase of the sulfur-containing amino acid homocysteine in plasma-called hyperhomocysteinemia-is associated with a high risk of migraine, especially migraine with aura. In animal studies, rats with hyperhomocysteinemia demonstrated mechanical allodynia, photophobia, and anxiety, and higher sensitivity to cortical spreading depression. In addition, rats with hyperhomocysteinemia were more sensitive in a model of chronic migraine induced by nitroglycerin which indicated the involvement of peripheral nociceptive mechanisms. The present work aimed to analyze the excitability of meningeal afferents and neurons isolated from the trigeminal ganglion of rats with prenatal hyperhomocysteinemia. Experiments were performed on male rats born from females fed with a methionine-rich diet before and during pregnancy. The activity of meningeal afferents was recorded extracellularly in hemiskull preparations exvivo and action potentials were characterized using cluster analysis. The excitability of trigeminal ganglion neurons was assessed using whole-cell patch clamp recording techniques and calcium imaging studies. Meningeal mast cells were stained using toluidine blue. The baseline extracellular recorded electrical activity of the trigeminal nerve was higher in the hyperhomocysteinemia group with larger amplitude action potentials. Lower concentrations of KCl caused an increase in the frequency of action potentials of trigeminal afferents recorded in rat hemiskull exvivo preparations. In trigeminal ganglion neurons of rats with hyperhomocysteinemia, the current required to elicit at least one action potential (rheobase) was lower, and more action potentials were induced in response to stimulus of 2 × rheobase. In controls, short-term application of homocysteine and its derivatives increased the frequency of action potentials of the trigeminal nerve and induced Ca2+ transients in neurons, which are associated with the activation of NMDA receptors. At the same time, in rats with hyperhomocysteinemia, we did not observe an increased response of the trigeminal nerve to NMDA. Similarly, the parameters of Ca2+ transients induced by NMDA, homocysteine, and its derivatives were not changed in rats with hyperhomocysteinemia. Acute incubation of the meninges in homocysteine and homocysteinic acid did not change the state of the mast cells, whereas in the model of hyperhomocysteinemia, an increased degranulation of mast cells in the meninges was observed. Our results demonstrated higher excitability of the trigeminal system of rats with hyperhomocysteinemia. Together with our previous finding about the lower threshold of generation of cortical spreading depression in rats with hyperhomocysteinemia, the present data provide evidence of homocysteine as a factor that increases the sensitivity of the peripheral migraine mechanisms, and the control of homocysteine level may be an important strategy for reducing the risk and/or severity of migraine headache attacks.

Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

Cannabinoid CB1 Receptor Expression and Localization in the Dorsal Horn of Male and Female Rat and Human Spinal Cord

ABSTRACT Background Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species. Aims We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans. Methods To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets. Results We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans. Conclusions The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.

The Effects of Mutual Interaction of Orexin-A and Glucagon-Like Peptide-1 on Reflex Swallowing Induced by SLN Afferents in Rats.

(1) Background: Our previous studies revealed that orexin-A, an appetite-increasing peptide, suppressed reflex swallowing via the commissural part of the nucleus tractus solitarius (cNTS), and that glucagon-like peptide-1 (GLP-1), an appetite-reducing peptide, also suppressed reflex swallowing via the medial nucleus of the NTS (mNTS). In this study, we examined the mutual interaction between orexin-A and GLP-1 in reflex swallowing. (2) Methods: Sprague–Dawley rats under urethane–chloralose anesthesia were used. Swallowing was induced by electrical stimulation of the superior laryngeal nerve (SLN) and was identified by the electromyographic (EMG) signals obtained from the mylohyoid muscle. (3) Results: The injection of GLP-1 (20 pmol) into the mNTS reduced the swallowing frequency and extended the latency of the first swallow. These suppressive effects of GLP-1 were not observed after the fourth ventricular administration of orexin-A. After the injection of an orexin-1 receptor antagonist (SB334867) into the cNTS, an ineffective dose of GLP-1 (6 pmol) into the mNTS suppressed reflex swallowing. Similarly, the suppressive effects of orexin-A (1 nmol) were not observed after the injection of GLP-1 (6 pmol) into the mNTS. After the administration of a GLP-1 receptor antagonist (exendin-4(5-39)), an ineffective dose of orexin-A (0.3 nmol) suppressed reflex swallowing. (4) Conclusions: The presence of reciprocal inhibitory connections between GLP-1 receptive neurons and orexin-A receptive neurons in the NTS was strongly suggested.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

Electromyography and Fos immunostaining study establish a possible functional link between trigeminal proprioception and the oculomotor system in rats.

The objective of this study was to explore whether there was a functional link between trigeminal proprioception and the oculomotor system mediated through jaw muscle afferents. Electromyography (EMG) was undertaken of the levator palpebrae (LP) and superior rectus (SR), and Fos expression was detected in the brainstem following consecutive down-stretching of the lower jaw at 2-4 Hz in rats. Retrograde tracing was undertaken of the interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN) pre-oculomotor neurons. EMG-like responses were recorded from the LP/SR during down-stretching of the lower jaw at 2-4 Hz in 3 out of 11 rats. Fos expression was induced by consecutive down-stretching of the lower jaw at 2-4 Hz for 20-30 seconds. Interestingly, Fos expression was distributed mainly in the bilateral INC/DN area. We also examined Fos-like immunoreactivity in central mesencephalic and paramedian pontine reticular formation that harbors premotor neurons controlling horizontal eye movement, but no Fos-like staining was observed therein. By injection of retrograde tracers into the oculomotor nucleus combined with Fos immunostaining, double labeled pre-oculomotor neurons were visualized to distribute in the INC/DN. In conclusions, there may exist a trigeminal proprioceptive – oculomotor system neural circuit through jaw muscle afferents in rats. Judging from Fos distribution pattern, this pathway might be related to vertical and torsional eye movements.

TRPA1 mediates amplified sympathetic responsiveness to activation of metabolically sensitive muscle afferents in rats with femoral artery occlusion

Autonomic responses to activation of mechanically and metabolically sensitive muscle afferent nerves during static contraction are augmented in rats with femoral artery occlusion. Moreover, metabolically sensitive transient receptor potential cation channel subfamily A, member 1 (TRPA1) has been reported to contribute to sympathetic nerve activity (SNA) and arterial blood pressure (BP) responses evoked by static muscle contraction. Thus, in the present study, we examined the mechanisms by which afferent nerves' TRPA1 plays a role in regulating amplified sympathetic responsiveness due to a restriction of blood flow directed to the hindlimb muscles. Our data show that 24–72 h of femoral artery occlusion (1) upregulates the protein levels of TRPA1 in dorsal root ganglion (DRG) tissues; (2) selectively increases expression of TRPA1 in DRG neurons supplying metabolically sensitive afferent nerves of C-fiber (group IV); and (3) enhances renal SNA and BP responses to AITC (a TRPA1 agonist) injected into the hindlimb muscles. In addition, our data demonstrate that blocking TRPA1 attenuates SNA and BP responses during muscle contraction to a greater degree in ligated rats than those responses in control rats. In contrast, blocking TRPA1 fails to attenuate SNA and BP responses during passive tendon stretch in both groups. Overall, results of this study indicate that alternations in muscle afferent nerves' TRPA1 likely contribute to enhanced sympathetically mediated autonomic responses via the metabolic component of the muscle reflex under circumstances of chronic muscle ischemia.

Microsecond-scale timing precision in rodent trigeminal primary afferents.

Communication in the nervous system occurs by spikes: the timing precision with which spikes are fired is a fundamental limit on neural information processing. In sensory systems, spike-timing precision is constrained by first-order neurons. We found that spike-timing precision of trigeminal primary afferents in rats and mice is limited both by stimulus speed and by electrophysiological sampling rate. High-speed video of behaving mice revealed whisker velocities of at least 17,000°/s, so we delivered an ultrafast "ping" (>50,000°/s) to single whiskers and sampled primary afferent activity at 500 kHz. Median spike jitter was 17.4 μs; 29% of neurons had spike jitter < 10 μs. These results indicate that the input stage of the trigeminal pathway has extraordinary spike-timing precision and very high potential information capacity. This timing precision ranks among the highest in biology.

ATP decreases mechanical sensitivity of muscle thin-fiber afferents in rats

ATP is an energy rich substance contained in cells in the order of mM. It is released when cells are damaged and when muscle is compressed or contracted. Subcutaneous injection of ATP induces pain-related behavior and hyperalgesia to mechanical and heat stimulation in rats. However, the effects of ATP in muscle have not been fully studied. In the present study we examined the effects of ATP on muscle C-fiber afferent activities using single fiber recordings, and on nociceptive behavior. Muscle C-fiber activities were recorded in vitro using extensor digitorum longus muscle–common peroneal nerve preparations excised from rats deeply anesthetized with pentobarbital. ATP (100μM and 1mM, but not 1μM) superfused for 5min before the mechanical stimulation suppressed the mechanical responses of muscle thin fibers irrespective of whether they excited the fiber. This suppressive effect was reversed by P2X receptor antagonists PPADS (100μM) and suramin (300μM). We also found that subcutaneous injection of ATP (10mM) induced nociceptive behavior, whereas intramuscular injection had no effect. These findings showed that effects of ATP on muscle afferents differ from those on cutaneous afferents.

Inhibition of Peripheral FAAH Depresses Activities of Bladder Mechanosensitive Nerve Fibers of the Rat

FAAH degrades endocannabinoids and fatty acid amides. FAAH inhibition reduces micturition frequency and counteracts bladder overactivity in rats. We studied the effects of the peripherally active selective FAAH inhibitor URB937, and the CB1 and CB2 receptor antagonists rimonabant and SR144528, respectively, on single unit afferent activity of primary bladder afferents in rats. Female Sprague Dawley® rats were anesthetized. Single unit afferent activity of Aδ or C-fibers from the L6 dorsal roots was recorded during bladder filling before and after URB937 administration with or without rimonabant or SR144528. Drugs (1 mg/kg) were given intravenously. FAAH, CB1 and CB2 expression, and expression of the sensory marker CGRP inthe L6 dorsal root ganglion were compared by immunofluorescence. A total of 102 single afferent fibers (48 Aδ and 54 C-fibers) were isolated from 57 rats. URB937 decreased single unit afferent activity of C-fibers to a mean ± SEM of 78% ± 9% and of Aδ-fibers to a mean of 67% ± 7% while increasing bladder compliance to a mean of 116% ± 3%. The effects of URB937 on single unit afferent activity and bladder compliance were counteracted by rimonabant or SR144528. Rimonabant increased single unit afferent activity ofeach fiber type but SR144528 affected only Aδ-fiber activity. CGRP positive L6dorsal root ganglion neurons showed strong FAAH, CB1 and CB2 staining. To our knowledge we report for the first time that inhibiting peripheral FAAH depresses the Aδ and C-fiber activity of primary bladder afferents via CB1 and CB2 receptors. CB antagonists alone exerted facilitatory effects on single unit afferent activity during bladder filling in rats. The endocannabinoid system may be involved in physiological control of micturition as regulators of afferent signals.

Sex differences in the ability of high fat diet to inhibit glucose‐induced modulation of 5‐HT3 receptor responses in GI‐vagal afferents in rats (905.4)

Previous studies in this lab have shown that extracellular glucose modulates 5‐HT3 receptor (5‐HT3R) mediated currents GI vagal afferent neurons and that this response is lost following exposure to a high fat diet (HFD). The aim of this study was to investigate whether this loss of glucose‐induced modulation showed any sex dependent differences.Sprague‐Dawley rats were placed on HFD (60% kcal from fat) at 28 days. Whole cell patch clamp recordings were made subsequently from acutely dissociated identified GI‐projecting vagal afferent neurons. The ability of extracellular glucose (2.5‐10mM) to modulate the magnitude of the 5‐HT‐induced inward current was assessed.After 4 weeks HFD exposure, glucose modulated the 5‐HT‐induced response in 7/8 neurons (87%) from female rats. In contrast, glucose modulated the 5‐HT‐induced inward current in only 4/7 neurons (57%; 4 weeks) from male rats (P&lt;0.2). At this time point, there was no difference in body weights (male: 189±12g vs female: 168±5g; P&gt;0.05) although male rats had significantly higher blood glucose levels (363±13mg/dl vs 251±7mg/dl; P&lt;0.05). These data suggest that, following HFD (i) male rats may be more susceptible to loss of glucose‐induced modulation of 5‐HT‐induced currents in GI vagal afferent neurons and (ii) this loss of glucose‐induced modulation is determined primarily by dysregulated glycemic control rather than diet or obesity.Grant Funding Source: NIH DK078364, NSF IOS1148978

Glial cell line‐derived neurotrophic factor sensitized the mechanical response of muscular thin‐fibre afferents in rats

The role of glial cell line-derived neurotrophic factor (GDNF) in pain and muscular nociceptor activities is not well understood. We examined pain-related behaviour and mechanical response of muscular thin-fibre afferents after intramuscular injection of GDNF in rats. GDNF and antagonist to transient receptor potential V1 or acid-sensing ion channels were injected into rat gastrocnemius muscle and muscular mechanical hyperalgesia was assessed with a Randall-Selitto analgesiometer. Activities of single C- (conduction velocity < 2.0 m/s) and Aδ-fibres (conduction velocity 2.0-12.0 m/s) were recorded from extensor digitorum longus muscle-nerve preparations in vitro. The changes in the responses to mechanical stimuli before and after GDNF injection were recorded. Mechanical hyperalgesia was observed from 1 h to 1 day after GDNF (0.03 μM, 20 μL) injection. The decreased withdrawal threshold was temporarily reversed after intramuscular injection of amiloride (50 mM, 20 μL), but not capsazepine (50 μM, 20 μL). In single-fibre recordings, both phosphate buffered saline (PBS) and GDNF failed to induce any significant discharges. GDNF significantly enhanced the mechanical response when compared with the PBS group, but only in Aδ-fibre afferents. C-fibres were not affected. Significantly lowered threshold and increased response magnitude to mechanical stimuli were observed 30 or 60-120 min after injection. These times are compatible with the timing of the onset of the hyperalgesic effect of GDNF. These results suggest that GDNF increased the response of muscular Aδ-fibre afferents to mechanical stimuli, resulting in muscular mechanical hyperalgesia.

Gastrointestinal pain: Unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation

The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3′,5′-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress–induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.

Inhibition of hydrogen sulfide restores normal breathing stability and improves autonomic control during experimental heart failure

Cardiovascular autonomic imbalance and breathing instability are major contributors to the progression of heart failure (CHF). Potentiation of the carotid body (CB) chemoreflex has been shown to contribute to these effects. Hydrogen sulfide (H2S) recently has been proposed to mediate CB hypoxic chemoreception. We hypothesized that H2S synthesis inhibition should decrease CB chemoreflex activation and improve breathing stability and autonomic function in CHF rats. Using the irreversible inhibitor of cystathione γ-lyase dl-propargylglycine (PAG), we tested the effects of H2S inhibition on resting breathing patterns, the hypoxic and hypercapnic ventilatory responses, and the hypoxic sensitivity of CB chemoreceptor afferents in rats with CHF. In addition, heart rate variability (HRV) and systolic blood pressure variability (SBPV) were calculated as an index of autonomic function. CHF rats, compared with sham rats, exhibited increased breath interval variability and number of apneas, enhanced CB afferent discharge and ventilatory responses to hypoxia, decreased HRV, and increased low-frequency SBPV. Remarkably, PAG treatment reduced the apnea index by 90%, reduced breath interval variability by 40-60%, and reversed the enhanced hypoxic CB afferent and chemoreflex responses observed in CHF rats. Furthermore, PAG treatment partially reversed the alterations in HRV and SBPV in CHF rats. Our results show that PAG treatment restores breathing stability and cardiac autonomic function and reduces the enhanced ventilatory and CB chemosensory responses to hypoxia in CHF rats. These results support the idea that PAG treatment could potentially represent a novel pathway to control sympathetic outflow and breathing instability in CHF.

Role for NGF in augmented sympathetic nerve response to activation of mechanically and metabolically sensitive muscle afferents in rats with femoral artery occlusion

Arterial blood pressure and heart rate responses to static contraction of the hindlimb muscles are greater in rats whose femoral arteries were previously ligated than in control rats. Also, the prior findings demonstrate that nerve growth factor (NGF) is increased in sensory neurons-dorsal root ganglion (DRG) neurons of occluded rats. However, the role for endogenous NGF in engagement of the augmented sympathetic and pressor responses to stimulation of mechanically and/or metabolically sensitive muscle afferent nerves during static contraction after femoral artery ligation has not been specifically determined. In the present study, both afferent nerves and either of them were activated by muscle contraction, passive tendon stretch, and arterial injection of lactic acid into the hindlimb muscles. Data showed that femoral occlusion-augmented blood pressure response to contraction was significantly attenuated by a prior administration of the NGF antibody (NGF-Ab) into the hindlimb muscles. The effects of NGF neutralization were not seen when the sympathetic nerve and pressor responses were evoked by stimulation of mechanically sensitive muscle afferent nerves with tendon stretch in occluded rats. In addition, chemically sensitive muscle afferent nerves were stimulated by lactic acid injected into arterial blood supply of the hindlimb muscles after the prior NGF-Ab, demonstrating that the reflex muscle responses to lactic acid were significantly attenuated. The results of this study further showed that NGF-Ab attenuated an increase in acid-sensing ion channel subtype 3 (ASIC3) of DRG in occluded rats. Moreover, immunohistochemistry was employed to examine the number of C-fiber and A-fiber DRG neurons. The data showed that distribution of DRG neurons with different thin fiber phenotypes was not notably altered when NGF was infused into the hindlimb muscles. However, NGF increased expression of ASIC3 in DRG neurons with C-fiber but not A-fiber. Overall, these data suggest that 1) NGF is amplified in sensory nerves of occluded rats and contributes to augmented reflex sympathetic and blood pressure responses evoked by stimulation of chemically, but not mechanically, sensitive muscle afferent nerves and 2) NGF likely plays a role in modulating the muscle metaboreflex via enhancement of ASIC3 expression in C-fiber of DRG neurons.

Neutralization of NGF Attenuates Augmented Sympathetic Nerve Responses to Activation of Chemically Sensitive Muscle Afferents in Rats with Femoral Artery Occlusion

Arterial blood pressure and heart rate responses to static contraction of the hindlimb muscles are greater in rats whose femoral artery is previously ligated than in control rats. Also, the prior findings demonstrate that nerve growth factor (NGF) is amplified in dorsal root ganglion (DRG) neurons of occluded rats. NGF has been reported to sensitize responsiveness to mechanical and chemical stimuli via thin fiber afferent nerves. Thus, the present study was first to examine the role for NGF in engagement of the augmented sympathetic and pressor responses to muscle contraction and passive tendon stretch after 24 hrs of femoral artery ligation. The data showed that femoral occlusion‐augmented blood pressure response to contraction was significantly attenuated by the prior administration of NGF antibody into the hindlimb muscles. The effects of NGF neutralization were not seen as the sympathetic and pressor responses were evoked by stimulation of muscle mechanically sensitive afferent nerves with muscle stretch in occluded rats. In addition, chemically sensitive afferent nerves were stimulated by lactic acid injected into the arterial blood supply of the hindlimb muscles after the prior injection of NGF antibody, demonstrating that the reflex responses to lactic acid were significantly attenuated. Moreover, immunohistochemistry was employed to examine the number of DRG neurons that project C‐fiber and A‐fiber. The data showed that distribution of DRG neurons with thin fiber phenotypes was not notably altered as NGF was infused into the hindlimb muscles. However, NGF increased expression of acid sensing ion channel subtype 3 in DRG neurons with thin C‐fiber, but not A‐fiber. Overall, these data suggest that NGF increased in sensory nerves of occluded rats is likely engaged in augmented reflex sympathetic and blood pressure responses evoked by stimulation of chemically, but not mechanically sensitive muscle afferent nerves.

Intravenous ascorbic acid does not block the pressor or sympathetic nerve response to ischemic fatiguing rhythmic handgrip exercise

Reactive oxygen species stimulate group IV muscle afferents in rats but the effects of oxidative stress on the exercise pressor reflex in humans is unknown. The purpose of the current study was to determine the effect of intravenous ascorbic acid (Vitamin C) on the exercise pressor reflex in humans. Seven young healthy nonsmokers participated in the current study, which was part of a large protocol in our laboratory. On Day 1, they underwent systemic infusions of saline and then Vitamin C; on Day 2, they underwent a saline time/volume control. Muscle sympathetic nerve activity (MSNA), heart rate, and blood pressure were monitored throughout baseline, ischemic fatiguing rhythmic handgrip exercise, and post‐handgrip circulatory arrest. The change in mean arterial pressure from baseline to fatigue was not different between saline (Δ 35±6 mmHg) and Vitamin C (Δ 31±6 mmHg). Similarly, heart rate (Δ 22±4 bpm vs Δ 26±4 bpm) and MSNA burst rate (Δ 22±6 bpm vs Δ 22±5bpm) were also not different between treatments. Metaboreflex activation and time control experiments similarly showed no treatment effects. The current data suggest that oxidative stress does not contribute to the magnitude of blood pressure or MSNA response seen during fatiguing handgrip in young healthy subjects.This study supported by P01 HL096570 (LIS) and UL1 RR033184 (LIS)

Effects of TRPV4 cation channel activation on the primary bladder afferent activities of the rat

Transient receptor potential vanilloid 4 (TRPV4) may affect afferent pathways innervating the bladder. We investigated the effects of GSK1016790A (GSK) and RN1734, a TRPV4 agonist and antagonist, respectively, and P2X-purinoceptor antagonists (TNP-ATP and PPADS) on cystometry (CMG), and the effect of GSK on single afferent fiber activities (SAAs) of the rat bladder and its relationship with capsaicin (Cap)-sensitivity. Conscious female Sprague-Dawley rats were used for CMG measurements. In SAA measurements, under urethane anesthesia, SAA was identified by electrical stimulation of the pelvic nerve and by bladder distention. Cystometric parameters were measured before and after intravesical drug instillation. In SAA measurements, response with saline instillation served as baseline. Then, GSK was instilled three times, and finally Cap was instilled to investigate the relationship with Cap-sensitivity. Intravesical GSK-instillation transiently decreased bladder capacity and voided volume, which were counteracted by RN1734, TNP-ATP, and PPADS. In SAA measurements, Aδ-fibers (n = 7) were not affected by either GSK or Cap. Based on the Cap-sensitivity, C-fibers could be divided into two subtypes: Cap-insensitive (n = 14) and Cap-sensitive (n = 8). In the Cap-insensitive C-fibers, GSK significantly increased the SAAs during the first instillation, but the increase attenuated with time, whereas GSK did not significantly affect the Cap-sensitive C-fibers. The present results suggest that activation of TRPV4 in the bladder, probably urothelium, facilitates the micturition reflex by activation of the mechanosensitive, Cap-insensitive C-fibers of the primary bladder afferents in rats.

Bilateral pudendal afferent stimulation improves bladder emptying in rats with urinary retention

To determine whether bilateral electrical stimulation (BiES) of the transected pudendal sensory nerves could further enhance the voiding efficiency beyond that produced by unilateral electrical stimulation (UniES) of transected pudendal afferents in rats with urinary retention. The efficiency of bladder emptying with either UniES or BiES of pudendal nerve afferents was measured after acute bilateral transection of the sensory branch of the pudendal nerve. The effects of UniES and BiES on voiding in a partially denervated bladder and acute spinal transection, respectively, were determined. The voiding efficiency (VE) was reduced from 69 to 22% after bilateral transection of the sensory branch of the pudendal nerve. UniES or BiES increased the VE to 49-62%. Although in most instances BiES consistently generated more efficient bladder emptying than did UniES, these differences were not significant. Both UniES and BiES increased VE after unilateral pelvic nerve transection, demonstrating efficacy in a partially denervated bladder. The enhancement of VE by either UniES or BiES was preserved after acute T(9)-T(10), demonstrating the spinal origin of this augmenting reflex. The results of the present study are consistent with an essential role for pudendal sensory feedback in efficient bladder emptying, and unilateral and bilateral electrical activation of pudendal nerve afferents are equally efficient in improving bladder emptying in an animal model of urinary retention. This could provide an approach to improve bladder emptying in patients with non-obstructive urinary retention.