Decerebrate Rats Research Articles

GsMTx4 Reduces the Pressor Response during Dynamic Hindlimb Muscle Stretch in Decerebrate Rats

The exercise pressor reflex, which is activated by mechanical and metabolic stimuli arising from within contracting skeletal muscles, contributes to the increase in blood pressure that occurs during exercise. GsMTx4, a mechano‐gated channel inhibitor that is partially selective for piezo channels, was recently found to reduce the pressor response during static rat hindlimb muscle stretch, a maneuver that is commonly used to study the mechanical component of the exercise pressor reflex. However, the GsMTx4‐induced reduction of the pressor response was limited to the initial phase (i.e., the first ~5 sec) of the stretch when muscle length was changing. That finding may reflect the rapid deactivation kinetics of piezo2 channels and the possibility that they do not contribute importantly to the later phases of a static stretch when muscle length is not changing. To investigate this possibility, we tested the hypothesis that in decerebrate, unanesthetized rats, GsMTx4 would reduce the pressor response throughout the duration of a 1 Hz dynamic hindlimb muscle stretch protocol that produced repeated changes in muscle length. Young adult male Sprague‐Dawley rats (n=12) had a jugular vein and both common carotid arteries cannulated. The left calcaneus was cut and the triceps surae muscles were linked by string to a force transducer. The pressor response during 30 seconds of 1 Hz dynamic stretch was measured before and after the injection of 10 μg of GsMTx4 into the arterial supply of the left hindlimb. We found that GsMTx4 reduced the peak pressor response during dynamic stretch (control: 15±4, GsMTx4: 5±2 mmHg, p=0.047, n=7). Moreover, the effect of GsMTx4 on the pressor response was evident throughout the duration of the dynamic stretch protocol. GsMTx4 did not, however, reduce the pressor response that resulted from the hindlimb arterial injection of 24 mmol lactic acid (control: 32±5, post‐GsMTx4: 29±4 mmHg, p=0.53) which indicates that GsMTx4 did not block voltage‐gated sodium channels in our experiments. Furthermore, the injection of GsMTx4 into the jugular vein (n=5) had no effect on the pressor response during dynamic stretch (control: 19±6, GsMTx4: 19±4, p=0.98). This finding indicates that when GsMTx4 was injected into the arterial supply of the hindlimb its effect on the pressor response during stretch was due to its actions on the peripheral endings of the sensory neurons. Our present findings suggest that piezo channels, most likely piezo2 channels, contribute to the pressor response throughout the duration of dynamic hindlimb muscle stretch in decerebrate rats.Support or Funding InformationSupported by university/departmental fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blood pressure responses to hindlimb arterial bradykinin injection are mediated by bradykinin 2 receptors in decerebrate rats

Mechanical and metabolic signals associated with skeletal muscle contraction activate a reflex that increases blood pressure during exercise (i.e., the exercise pressor reflex). Bradykinin is one metabolite that has been found to play a role in activating the exercise pressor reflex and also contribute to the reflex's exaggeration in a rat model of simulated peripheral artery disease (PAD) induced by chronic femoral artery ligation. Specifically, bradykinin 2 (B2) receptor expression was found to be greater in rat dorsal root ganglia (DRG) tissue of “ligated” rats compared to the expression found in DRG tissue of rats in which the femoral artery was patent (i.e., “freely perfused”). Moreover, the hindlimb arterial injection of bradykinin reflexly increased blood pressure more in ligated rats than it did in freely perfused rats. We tested the hypotheses that, 1) HOE‐140 (5 μg/kg), a selective B2 receptor antagonist, would reduce the pressor response to bradykinin (0.5 μg/kg) injection in both ligated and freely perfused decerebrate, unanesthetized rats and 2) the magnitude of the reduction would be greater in ligated rats than in freely perfused rats. In five rats in which a femoral artery was ligated 72 hours before the experiment, HOE‐140 significantly reduced the pressor response to bradykinin injection (control: 23±2, post HOE‐140: 5±1 mmHg; p<0.01). In freely perfused rats, bradykinin injection evoked pressor responses in two rats and depressor responses in four rats. HOE‐140 reduced the magnitude of the pressor response (control: 16±7, HOE‐140: 4±2 mmHg, n=2) and the depressor response (pre: −22±5, post: −2±1 mmHg; p=0.02, n=4) in those experiments. We hypothesized that the depressor response in the freely perfused rats was due to the fact that hindlimb blood flow was not arrested during bradykinin injection and, therefore, the vasodilatory effects of bradykinin overwhelmed reflex‐mediated vasoconstriction. In four additional freely perfused rats in which a depressor response to bradykinin injection was initially observed (−31±8 mmHg), we subsequently tightened an iliac artery/vein snare to arrest the hindlimb circulation. Tightening the snare in this manner unmasked bradykinin injection‐induced pressor responses (9±4 mmHg). We conclude that hindlimb arterial bradykinin injection in ligated rats consistently produced a pressor response that was mediated by B2 receptors. In freely perfused rats, the divergent responses to bradykinin injection (i.e., pressor or depressor) were also B2 receptor mediated. B2 receptors may play a role in evoking the exaggerated exercise pressor reflex that is found in the rat model of simulated PAD.Support or Funding InformationSupported by University/Department funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isoflurane anesthesia does not affect spinal cord neurovascular coupling: evidence from decerebrated rats.

Neurological examination remains the primary clinical investigation in patients with spinal cord injury. However, neuroimaging methods such as functional magnetic resonance imaging (fMRI) are promising tools for following functional changes in the course of injury, disease and rehabilitation. However, the relationship between neuronal activity and blood flow in the spinal cord on which fMRI relies has been largely overlooked. The objective of this study was to examine neurovascular coupling in the spinal cord of decerebrated rats during electrical stimulation of the sciatic nerve with and without isoflurane anesthesia (1.2%). Local field potentials (LFP) and spinal cord blood flow (SCBF) were recorded simultaneously in the lumbosacral enlargement. Isoflurane did not significantly alter LFP (p = 0.53) and SCBF (p = 0.57) amplitude. Accordingly, neurovascular coupling remained comparable with or without isoflurane anesthesia (p = 0.39). These results support the use of isoflurane in rodents to investigate nociceptive functions of the spinal cord using fMRI.

µ-Opioid receptors inhibit the exercise pressor reflex by closing N-type calcium channels but not by opening GIRK channels in rats.

µ-Opioid G protein-coupled receptors (MOR) interact with ion channels to decrease neuronal excitability. In humans, intrathecal administration of the MOR agonist fentanyl inhibits the exercise pressor reflex, an effect that can be attributed to either the opening of inward rectifying potassium channels (GIRK) or the closing of N-type calcium channels. The purpose of this study was to determine if the highly selective MOR agonist [d-Ala2, N-MePhe4,Gly-ol]-enkephalin (DAMGO) attenuates the exercise pressor reflex and which of these two channels are responsible for this effect. In decerebrate rats, we determined the effect of intrathecal injection of either tertiapin-LQ, which blocks the GIRK channel or ω-conotoxin-GVIA, which blocks the N-type calcium channel on the exercise pressor reflex, which was evoked by contracting the triceps surae muscles. Initially, we established that intrathecal injection of DAMGO inhibited the exercise pressor reflex relative to no intrathecal injection or intrathecal saline injection ( P < 0.001, n = 5). We then found that intrathecal injection of two doses of tertiapin-LQ (1 and 10 µg) had no effect on the exercise pressor reflex ( n = 6 and n = 7, respectively; P > 0.05). Importantly, neither dose of tertiapin-LQ prevented the DAMGO-induced inhibition of the exercise pressor reflex. Last, we found that intrathecal injection of ω-conotoxin-GVIA markedly attenuated the exercise pressor reflex ( P < 0.001, n = 7). The cardioaccelerator response to contraction did not appear to be effected in any of the experiments. We conclude that N-type voltage-gated calcium channel inhibition appears to be the mechanism by which MOR activation inhibits the exercise pressor reflex in decerebrate rats.

Phrenic motoneurons: output elements of a highly organized intraspinal network.

pontomedullary respiratory network generates the respiratory pattern and relays it to bulbar and spinal respiratory motor outputs. The phrenic motor system controlling diaphragm contraction receives and processes descending commands to produce orderly, synchronous, and cycle-to-cycle-reproducible spatiotemporal firing. Multiple investigators have studied phrenic motoneurons (PhMNs) in an attempt to shed light on local mechanisms underlying phrenic pattern formation. I and colleagues (Marchenko V, Ghali MG, Rogers RF. Am J Physiol Regul Integr Comp Physiol 308: R916-R926, 2015.) recorded PhMNs in unanesthetized, decerebrate rats and related their activity to simultaneous phrenic nerve (PhN) activity by creating a time-frequency representation of PhMN-PhN power and coherence. On the basis of their temporal firing patterns and relationship to PhN activity, we categorized PhMNs into three classes, each of which emerges as a result of intrinsic biophysical and network properties and organizes the orderly contraction of diaphragm motor fibers. For example, early inspiratory diaphragmatic activation by the early coherent burst generated by high-frequency PhMNs may be necessary to prime it to overcome its initial inertia. We have also demonstrated the existence of a prominent role for local intraspinal inhibitory mechanisms in shaping phrenic pattern formation. The objective of this review is to relate and synthesize recent findings with those of previous studies with the aim of demonstrating that the phrenic nucleus is a region of active local processing, rather than a passive relay of descending inputs.

Bradykinin does not acutely sensitize the reflex pressor response during hindlimb skeletal muscle stretch in decerebrate rats.

Hindlimb skeletal muscle stretch (i.e., selective activation of the muscle mechanoreflex) in decerebrate rats evokes reflex increases in blood pressure and sympathetic nerve activity. Bradykinin has been found to sensitize mechanogated channels through a bradykinin B2 receptor-dependent mechanism. Moreover, bradykinin B2 receptor expression on sensory neurons is increased following chronic femoral artery ligation in the rat (a model of simulated peripheral artery disease). We tested the hypothesis that injection of bradykinin into the arterial supply of a hindlimb in decerebrate, unanesthetized rats would acutely augment (i.e., sensitize) the increase in blood pressure and renal sympathetic nerve activity during hindlimb muscle stretch to a greater extent in rats with a ligated femoral artery than in rats with a freely perfused femoral artery. The pressor response during static hindlimb muscle stretch was compared before and after hindlimb arterial injection of 0.5 µg of bradykinin. Injection of bradykinin increased blood pressure to a greater extent in "ligated" (n = 10) than "freely perfused" (n = 10) rats. The increase in blood pressure during hindlimb muscle stretch, however, was not different before vs. after bradykinin injection in freely perfused (14 ± 2 and 15 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.62) or ligated (15 ± 3 and 14 ± 2 mmHg for pre- and post-bradykinin, respectively, P = 0.80) rats. Likewise, the increase in renal sympathetic nerve activity during stretch was not different before vs. after bradykinin injection in either group of rats. We conclude that bradykinin did not acutely sensitize the pressor response during hindlimb skeletal muscle stretch in freely perfused or ligated decerebrate rats.

Mechanism of physical stress-induced inhibition of ovarian estradiol secretion in anesthetized rats.

This study examined the site of main integration center in the physical stress-induced inhibition of ovarian estradiol secretion because of ovarian sympathetic nerve (superior ovarian nerve: SON) activation in anesthetized rats. In central nervous system-intact rats, electrical stimulation of the tibial afferent nerve at 10V increased the efferent activity of the SON by 39±13% and reduced the ovarian secretion of estradiol by 34±7%. These responses were observed in decerebrate rats but were abolished in spinal rats. Thus, the main integration center for this ovarian hormonal response is located in the brain stem.

The role played by oxidative stress in evoking the exercise pressor reflex in health and simulated peripheral artery disease.

Ligating the femoral artery of a rat for 72h, a model for peripheral artery disease, causes an exaggerated exercise pressor reflex in response to muscle contraction. Likewise, the hindlimb muscles of rats with ligated femoral arteries show increased levels of reactive oxygen species. Infusion of tiron, a superoxide scavenger, attenuated the exaggerated pressor reflex and reduced reactive oxygen species production in rats with ligated femoral arteries. Conversely, we found no effect of tiron infusion on the pressor reflex in rats with patent femoral arteries. These results suggest a role of reactive oxygen species with respect to causing the exaggerated pressor response to contraction seen in rats with ligated arteries and peripheral artery disease. Contraction of muscle evokes the exercise pressor reflex (EPR), which is expressed partly by increases in heart rate and arterial pressure. Patients with peripheral artery disease (PAD) show an exaggerated EPR, sometimes report pain when walking and are at risk for cardiac arrthymias. Previous research suggested that reactive oxygen species (ROS) mediate the exaggerated EPR associated with PAD. To examine the effects of ROS on the EPR, we infused a superoxide scavenger, tiron, into the superficial epigastric artery of decerebrated rats. In some, we simulated PAD by ligating a femoral artery for 72h before the experiment. The peak EPR in 'ligated' rats during saline infusion averaged 31±4mmHg, whereas the peak EPR in these rats during tiron infusion averaged 13±2mmHg (n=12; P<0.001); the attenuating effect of tiron on the EPR was partly reversed when saline was reinfused into the superficial epigastric artery (21±2 mmHg; P<0.01 vs. tiron). The peak EPR in 'ligated' rats was also attenuated (n=7; P<0.01) by infusion of gp91ds-tat, a peptide that blocks the activity of NAD(P)H oxidase. Tiron infusion had no effect on the EPR in rats with patent femoral arteries (n=9). Western blots showed that the triceps surae muscles of 'ligated' rats expressed more Nox2 and p67phox, which are components of NADPH oxidase, compared to triceps surae muscles of 'freely perfused' rats. Tiron added to muscle homogenates reduced ROS production in vitro. The results of the present study provide further evidence indicating that ROS mediates the exaggeration of EPR in rats with simulated PAD.

Chronic Femoral Artery Ligation Exaggerates the Exercise Pressor Reflex but Not Selective Activation of the Muscle Mechanoreflex in Decerebrate Rats

Mechanical and metabolic stimuli arising within contracting skeletal muscles reflexly increase sympathetic nervous system activity, blood pressure, and heart rate. This reflex, termed the exercise pressor reflex, is exaggerated in a rat model of limb ischemia in which a femoral artery is chronically ligated. We recently found that the mechanically‐sensitive component of the exercise pressor reflex contributes to its exaggeration in rats with a ligated femoral artery despite no difference in mechano‐gated channel protein expression in lumbar dorsal root ganglia (DRG) tissue. This suggests that metabolite‐induced sensitization of mechanoreceptors underlies the exaggerated mechanically‐sensitive component of the exercise pressor reflex in ligated rats. This prompted us to seek the physiological evidence in support of the anatomical evidence that ligation does not increase mechano‐gated channel expression in DRG tissue.ObjectiveWe used a within‐rat comparison experimental design to test the hypothesis that the increase in blood pressure, heart rate, and renal sympathetic nerve activity (RSNA) during selective activation of the muscle mechanoreflex (a purely mechanical stimulus) would not be different when evoked from a freely perfused hindlimb compared a ligated hindlimb.MethodsFifteen male Sprague‐Dawley rats had their left femoral artery ligated 72 hours before the experiment. In decerebrate, unanesthetized rats the muscle mechanoreflex was selectively activated by passively stretching the Achilles tendon for 30 s. In five of those fifteen rats, the exercise pressor reflex was evoked by electrically‐stimulating the sciatic nerves for 30 s which produced static hindlimb muscle contractions.ResultsIn 10 rats there were no differences between the peak pressor (freely perfused: 13±2, ligated: 14±3 mmHg, p=0.801), cardioaccelerator (freely perfused: 12±2, ligated: 11±2 bpm, p=0.672), or RSNA (freely perfused: 40±7, ligated: 46±6%, p=0.377) responses evoked by tendon stretch of the freely perfused and ligated hindlimbs. In five additional rats, the exercise pressor reflex was greater when evoked from the contracting ligated hindlimb (28±3 mmHg) compared to the contracting freely perfused hindlimb (21±3 mmHg, p=0.014). In those same five rats, however, in which femoral artery ligation exaggerated the exercise pressor reflex there was no difference in the peak pressor response to tendon stretch between hindlimbs (freely perfused: 16±2, ligated: 15±3 mmHg, p=0.581).ConclusionWe found that the reflex pressor, cardioaccelerator, and RSNA responses to tendon stretch (a purely mechanical stimulus) were not exaggerated by femoral artery ligation, even in rats in which the exercise pressor reflex was confirmed to be exaggerated by ligation. These findings indicate that the ligation‐induced exaggeration of the mechanically‐sensitive component of the exercise pressor reflex reported previously must be due to a metabolite‐induced sensitization of mechano‐gated receptors.Support or Funding InformationDepartmental funds

Effects of Type 1 Diabetes on the Exercise Pressor Reflex in Rats

Type 1 diabetes (T1DM) is known to cause both peripheral and autonomic neuropathy through mechanisms affecting thinly myelinated afferents. These same afferents may also play a role in the exercise pressor reflex suggesting that T1DM could alter this reflex as well. The purpose of this study was to establish the effects of type 1 diabetes on the pressor and cardioaccelerator responses evoked by statically contracting the hindlimb muscles at different time points of the disease and identify possible mechanisms responsible for these changes. We injected (i.p.) 50 mg/kg of Streptozotocin (STZ) or the vehicle (CTL) in either sex and waited 1wk (STZ: BW=267±12 g, glucose=438±24 mg/dL, HbA1C=6.5±0.3%; CTL: BW=328±16 g, glucose=167±10 mg/dL, HbA1C=4.4±0.1%), 3wks (STZ: BW=293±10 g, glucose=465±22 mg/dL, HbA1C=10±0.3%; CTL: BW=329±22 g, glucose=212±26 mg/dL, HbA1C=4.8±0.4%), or 6wks (STZ: BW=380±16 g, glucose=546±35 mg/dL, HbA1C=12±0.4%) before performing experiments. In unanaesthetized, decerebrate rats, we statically contracted the hindlimb muscles for 30s and measured changes in mean arterial pressure (MAP) and heart rate (HR). We then injected either α,β‐methylene ATP (20μg/kg; 200μl), a P2X receptor agonist, or lactic acid (24mM; 200μl), an ASIC3 channel agonist, into the arterial supply of the hindlimb and measured the evoked pressor and cardioaccelerator responses for each group. We found that the pressor (STZ: ΔMAP=25±5 mmHg, n=4; CTL: ΔMAP=13±2 mmHg, n=7; p=0.01) but not cardioaccelerator (STZ: ΔHR=23±9 bpm, n=4; CTL: ΔHR=13±3 bpm, n=7; p=0.10) response to static contraction was exaggerated 1wk after injecting STZ. Likewise, the pressor (STZ: ΔMAP=32±5 mmHg, n=8; CTL: ΔMAP=17±3 mmHg, n=6; p=0.02) but not cardioaccelerator (STZ: ΔHR=22±5 bpm, n=8; CTL: ΔHR=15±2 bpm, n=6; p=0.27) response to static contraction was exaggerated 3wks after injecting STZ. Conversely, both the pressor (6wks STZ: ΔMAP=5±2 mmHg, n=7) and cardioaccelerator (6wks STZ: ΔHR=1±2 bpm, n=7) responses to static contraction were significantly attenuated 6wks after injecting STZ compared to 1wk (MAP: p=0.02; HR: p=0.01) and 3wks after injecting STZ (MAP: p&lt;0.01; HR: p&lt;0.01). The developed tensions from contraction were similar within each comparison. Furthermore, we found that injecting lactic acid, but not α,β‐methylene ATP, into the arterial supply of the hindlimb evoked a greater pressor (STZ: ΔMAP=31±5 mmHg, n=9; CTL: ΔMAP=20±3 mmHg, n=13; p=0.04) and cardioaccelerator (STZ: ΔHR=13±3 bpm, n=9; CTL: ΔHR=3±2 bpm, n=13; p&lt;0.01) response 1wk after STZ injection compared to CTL. We conclude that the exercise pressor reflex is exaggerated in the early stages of T1DM but attenuated in the late stages of this disease. Additionally, ASIC3 channels but not P2X receptors may play a role in this exaggerated reflex 1wk after STZ injection.

The crossed phrenic phenomenon.

The cervical spine is the most common site of traumatic vertebral column injuries. Respiratory insufficiency constitutes a significant proportion of the morbidity burden and is the most common cause of mortality in these patients. In seeking to enhance our capacity to treat specifically the respiratory dysfunction following spinal cord injury, investigators have studied the “crossed phrenic phenomenon”, wherein contraction of a hemidiaphragm paralyzed by a complete hemisection of the ipsilateral cervical spinal cord above the phrenic nucleus can be induced by respiratory stressors and recovers spontaneously over time. Strengthening of latent contralateral projections to the phrenic nucleus and sprouting of new descending axons have been proposed as mechanisms contributing to the observed recovery. We have recently demonstrated recovery of spontaneous crossed phrenic activity occurring over minutes to hours in C1-hemisected unanesthetized decerebrate rats. The specific neurochemical and molecular pathways underlying crossed phrenic activity following injury require further clarification. A thorough understanding of these is necessary in order to develop targeted therapies for respiratory neurorehabilitation following spinal trauma. Animal studies provide preliminary evidence for the utility of neuropharmacological manipulation of serotonergic and adenosinergic pathways, nerve grafts, olfactory ensheathing cells, intraspinal microstimulation and a possible role for dorsal rhizotomy in recovering phrenic activity following spinal cord injury

Coordinated control of the tongue during suckling-like activity and respiration.

The tongue can move freely and is important in oral motor functions. Tongue movement must be coordinated with movement of the hyoid, mandible, and pharyngeal wall, to which it is attached. Our previous study using isolated brainstem-spinal cord preparations showed that application of N-methyl-D-aspartate induces rhythmic activity in the hypoglossal nerve that is coincident with rhythmic activity in the ipsilateral trigeminal motor nerve. Partial or complete midline transection of the preparation only abolishes activity in the trigeminal motor nerve; therefore, the neuronal network contributing to coordinated activity of the jaw/tongue muscles is located on both sides of the preparation and sends motor commands to contralateral trigeminal motoneurons. Arterially perfused decerebrate rat preparations exhibit stable inspiratory activity in the phrenic nerve, with efferent nerves innervating the upper airway muscles (the hypoglossal nerve, a branch of the cervical spinal nerve, the external branch of the superior laryngeal nerve, and the recurrent laryngeal nerve) under normocapnic conditions (5% CO2). During hypercapnia (8% CO2), pre-inspiratory discharges appear in all nerves innervating upper airway muscles. Such coordinated activity in the pre-inspiratory phase contributes to dilation of the upper airway and improves hypercapnia.

Coordinated Respiratory Motor Activity in Nerves Innervating the Upper Airway Muscles in Rats.

Maintaining the patency of the upper airway during breathing is of vital importance. The activity of various muscles is related to the patency of the upper airway. In the present study, we examined the respiratory motor activity in the efferent nerves innervating the upper airway muscles to determine the movements of the upper airway during respiration under normocapnic conditions (pH = 7.4) and in hypercapnic acidosis (pH = 7.2). Experiments were performed on arterially perfused decerebrate rats aged between postnatal days 21–35. We recorded the efferent nerve activity in a branch of the cervical spinal nerve innervating the infrahyoid muscles (CN), the hypoglossal nerve (HGN), the external branch of the superior laryngeal nerve (SLN), and the recurrent laryngeal nerve (RLN) with the phrenic nerve (PN). Inspiratory nerve discharges were observed in all these nerves under normocapnic conditions. The onset of inspiratory discharges in the CN and HGN was slightly prior to those in the SLN and RLN. When the CO2 concentration in the perfusate was increased from 5% to 8% to prepare for hypercapnic acidosis, the peak amplitudes of the inspiratory discharges in all the recorded nerves were increased. Moreover, hypercapnic acidosis induced pre-inspiratory discharges in the CN, HGN, SLN, and RLN. The onset of pre-inspiratory discharges in the CN, HGN, and SLN was prior to that of discharges in the RLN. These results suggest that the securing of the airway that occurs a certain time before dilation of the glottis may facilitate ventilation and improve hypercapnic acidosis.

Low-frequency stimulation of group III and IV hind limb afferents evokes reflex pressor responses in decerebrate rats.

Contraction of freely perfused hind limb muscles in decerebrate rats evokes the exercise pressor reflex, resulting in sympathetic activation and increased blood pressure. This reflex is propagated along mechanically sensitive group III and metabolically sensitive group IV afferent nerve fibers. Recent research by our laboratory has focused on the exaggeration of the exercise pressor reflex in decerebrate rats with simulated peripheral artery disease, which was induced by ligating the femoral artery for 72 h before the start of the experiment. Recently, we showed that ligating the femoral artery increased the responses of single fiber group III and IV triceps surae muscle afferents to static contraction. The objective of this study was to determine if electrical stimulation of group III and IV afferents at frequencies approximating those occurring during static contraction was capable of reflexively increasing arterial blood pressure. We directly stimulated muscle afferents in the absence of muscle contraction for both freely perfused and ligated rats. We established 0.25 Hz as the minimal stimulation frequency to observe a sustained blood pressure response. The blood pressure response increased in a graded fashion as both stimulus frequency and motor threshold were increased. Additionally, we observed similar blood pressure responses from both freely perfused and ligated rats, suggesting that spinal and medullary processing of group III and IV afferent input plays no role in augmenting the pressor response to contraction caused by femoral artery ligation.

Serotonin controls initiation of locomotion and afferent modulation of coordination via 5-HT7 receptors in adult rats.

Experiments on neonatal rodent spinal cord showed that serotonin (5-HT), acting via 5-HT7 receptors, is required for initiation of locomotion and for controlling the action of interneurons responsible for inter- and intralimb coordination, but the importance of the 5-HT system in adult locomotion is not clear. Blockade of spinal 5-HT7 receptors interfered with voluntary locomotion in adult rats and fictive locomotion in paralysed decerebrate rats with no afferent feedback, consistent with a requirement for activation of descending 5-HT neurons for production of locomotion. The direct control of coordinating interneurons by 5-HT7 receptors observed in neonatal animals was not found during fictive locomotion, revealing a developmental shift from direct control of locomotor interneurons in neonates to control of afferent input from the moving limb in adults. An understanding of the afferents controlled by 5-HT during locomotion is required for optimal use of rehabilitation therapies involving the use of serotonergic drugs. Serotonergic pathways to the spinal cord are implicated in the control of locomotion based on studies using serotonin type 7 (5-HT7 ) receptor agonists and antagonists and 5-HT7 receptor knockout mice. Blockade of these receptors is thought to interfere with the activity of coordinating interneurons, a conclusion derived primarily from in vitro studies on isolated spinal cord of neonatal rats and mice. Developmental changes in the effects of serotonin (5-HT) on spinal neurons have recently been described, and there is increasing data on control of sensory input by 5-HT7 receptors on dorsal root ganglion cells and/or dorsal horn neurons, leading us to determine the effects of 5-HT7 receptor blockade on voluntary overground locomotion and on locomotion without afferent input from the moving limb (fictive locomotion) in adult animals. Intrathecal injections of the selective 5-HT7 antagonist SB269970 in adult intact rats suppressed locomotion by partial paralysis of hindlimbs. This occurred without a direct effect on motoneurons as revealed by an investigation of reflex activity. The antagonist disrupted intra- and interlimb coordination during locomotion in all intact animals but not during fictive locomotion induced by stimulation of the mesencephalic locomotor region (MLR). MLR-evoked fictive locomotion was transiently blocked, then the amplitude and frequency of rhythmic activity were reduced by SB269970, consistent with the notion that the MLR activates 5-HT neurons, leading to excitation of central pattern generator neurons with 5-HT7 receptors. Effects on coordination in adults required the presence of afferent input, suggesting a switch to 5-HT7 receptor-mediated control of sensory pathways during development.

Effects of vagotomy on hypoglossal and phrenic responses to hypercapnia in the decerebrate rat

Hypercapnia characterizes a variety of physiological and pathological states and must be compensated effectively by the respiratory, cardiovascular, renal, and intra- and extracellular pH buffering systems to maintain homeostasis. Several studies have examined the respiratory response to hypercapnia, but contemporaneous changes in respiratory frequency and tidal volume prevent investigating the pure influence on respiratory amplitude. Therefore, we sought to test the effect of hypercapnia on hypoglossal (XII) and phrenic nerve (PN) inspiratory (Insp) and XII pre-inspiratory (pre-I) activities in vagus-intact and vagus-denervated animals. Experiments were performed on six artificially-ventilated unanesthetized pre-collicular decerebrate Sprague-Dawley adult male rats. Vagotomy under normocapnic conditions effected the consistent appearance of significant XII pre-I and a greater increase in XII than PN Insp amplitude. In the vagus-intact state, administration of a hypercapnic (5% CO2, 95% O2) gas mixture resulted in a greater increase in XII than PN Insp activity. In the vagotomized state, hypercapnia caused a drastic increase in XII pre-I and significant non-differential increases in both XII and PN Insp activity. The increase in XII pre-I was significantly greater than hypercapnia-induced increases in XII and PN Insp discharges. Following vagotomy, duration and amplitude of XII pre-I are potently modulated by CO2 tension. Based on our results, we conclude that vagal afferents exert differential inhibition of PN Insp and XII pre-I/Insp motor outputs. The role of vagal control in orchestration and optimization of respiratory response to hypercapnia is discussed.

Effects of Direct Afferent Nerve Stimulation Frequency and Intensity In Evoking the Exercise Pressor Reflex

Contraction of freely perfused hind limb muscles in decerebrate rats evokes the exercise pressor reflex, resulting in sympathetic activation and increased blood pressure. This reflex is primarily propagated along mechanically sensitive group III and metabolically sensitive group IV afferent nerve fibers. Recent research by our laboratory has focused on the exaggeration of the exercise pressor reflex in decerebrate rats with simulated peripheral artery disease, which was induced by ligating the femoral artery for 72 hours before the start of the experiment. Recently, we showed that ligating the femoral artery increased the responses of single fiber group III and IV triceps surae muscle afferents to static contraction. The objective of the present study was to determine if electrical stimulation of group III and IV afferents at frequencies approximating those occurring during static contraction was capable of reflexively increasing arterial blood pressure.In decerebrate unanesthetized rats, we first determined motor threshold by gradually increasing the current applied as a single pulse (0.01 ms) to the gastrocnemius nerve until a twitch was evoked from the triceps surae muscles. Once motor threshold was determined, we injected intravenously pancuronium bromide to paralyze the rat for the remainder of the experiment. Five minutes later, we stimulated the sciatic nerve for 30 seconds at either 5, 20, or 100 times motor threshold, currents that presumably recruited thick group III, thick and thin group III, and all group III and IV fibers, respectively. In addition, we stimulated (0.01 ms pulse duration) the gastrocnemius nerves with six different frequencies (0.1, 0.25, 0.5, 1.0, 3.0, or 5.0 Hz), applying them in random order. Particular attention was paid to frequencies of 0.25, 0.5 and 1.0 Hz because these represented the discharge rates displayed by group III and IV afferents responding to static contraction.Overall, we found that stimulation of the gastrocnemius nerve increased arterial blood pressure in a graded fashion as both stimulus frequency and motor threshold were increased. In particular, stimulation of the gastrocnemius nerve at 100 times motor threshold (n=7) significantly increased arterial blood pressure (MAP) over baseline levels at 0.25 Hz (Peak ΔMAP= 11 ± 2 mmHg, p&lt; 0.001), 0.5 Hz (Peak ΔMAP= 13 ± 3 mmHg, p&lt; 0.01), and 1.0 Hz (Peak ΔMAP= 18 ± 4 mmHg, p&lt; 0.01).In conclusion, we observed that afferent stimulation frequencies as low as 0.25 Hz are sufficient to increase arterial blood pressure. These findings are relevant, as previous studies from our lab have measured afferent responses to contraction in these frequency ranges. Additionally, we observed that increasing stimulus frequency resulted in greater increases in blood pressure, a finding that parallels our previous results in studies using ligated rats. As such, these and future studies provide valuable insight into the mechanisms underlying the exaggerated exercise pressor reflex we observe in an animal model of peripheral artery disease.Support or Funding InformationFunded by NIH R01 ‐ AR059397

The Mechano‐gated Channel Inhibitor GsMTx4 Reduces the Exercise Pressor Reflex in Decerebrate Rats

Mechanical and metabolic stimuli arising within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments to exercise. In cats and rats, gadolinium has been used to study the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano‐gated channels and may exert multiple off‐target effects. We tested the hypothesis that GsMTX4, a more selective mechano‐gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano‐gated Piezo channels, reduced the exercise pressor reflex. We measured the pressor, cardio accelerator, and renal sympathetic nerve responses to 30 seconds of Achilles tendon stretch and 30 seconds of electrically‐induced intermittent hindlimb muscle contraction before and after injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb of decerebrate, unanesthetized rats. In six rats, intra‐arterial injection of GsMTx4 reduced the peak pressor (control: 24±5, GsMTx4: 12±5 mmHg, p&lt;0.01), cardio accelerator and renal sympathetic nerve responses to tendon stretch, which is a purely mechanical stimulus. In contrast, GsMTx4 had no effect on the pressor responses to intra‐arterial injection of α,β‐methylene ATP (20 μg/kg, control: 19±5, GsMTx4: 20±5 mmHg, p=0.72) or lactic acid (24 mM, control: 49±7, GsMTx4: 53±11 mmHg, p=0.71), findings which indicated that GsMTx4 did not block metabolically‐sensitive channels. In eight rats, intra‐arterial injection of GsMTx4 reduced the peak pressor (control: 24±2, GsMTx4: 14±3 mmHg, p&lt;0.01), cardio accelerator, and renal sympathetic nerve responses to electrically‐induced intermittent hindlimb muscle contractions, which is a mixed mechanical and metabolic stimulus. In contrast to intra‐arterial injection, injection of GsMTx4 into the jugular vein had no effect on the pressor, cardio accelerator, or renal sympathetic nerve responses to either tendon stretch (n=5) or intermittent contraction (n=6), findings which indicated that GsMTx4 did not reduce the exercise pressor reflex by exerting effects within the central nervous system. In all experiments the developed tension and the tension‐time integrals were not different between control and GsMTx4 conditions (p&gt;0.05 for all). Quantitative RT‐PCR and Western blot analyses indicated that both classes of Piezo channel isoforms, namely Piezo1 and Piezo2, were natively expressed in rat dorsal root ganglia tissue (n=3). We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was likely attributable, at least in part, to its effect on mechano‐gated Piezo channels. Our findings may have important implications in conditions such as hypertension, heart failure, and peripheral artery disease in which the mechanically‐sensitive component of the exercise pressor reflex has been found to contribute to the exaggerated pressor responses to exercise seen in those pathophysiological states.Support or Funding InformationNIH HL‐096570 and AR‐059397

The mechano-gated channel inhibitor GsMTx4 reduces the exercise pressor reflex in decerebrate rats.

Mechanical and metabolic stimuli from contracting muscles evoke reflex increases in blood pressure, heart rate and sympathetic nerve activity. Little is known, however, about the nature of the mechano-gated channels on the thin fibre muscle afferents that contribute to evoke this reflex, termed the exercise pressor reflex. We determined the effect of GsMTx4, an inhibitor of mechano-gated Piezo channels, on the exercise pressor reflex evoked by intermittent contraction of the triceps surae muscles in decerebrated, unanaesthetized rats. GsMTx4 reduced the pressor, cardioaccelerator and renal sympathetic nerve responses to intermittent contraction but did not reduce the pressor responses to femoral arterial injection of compounds that stimulate the metabolically-sensitive thin fibre muscle afferents. Expression levels of Piezo2 channels were greater than Piezo1 channels in rat dorsal root ganglia. Our findings suggest that mechanically-sensitive Piezo proteins contribute to the generation of the mechanical component of the exercise pressor reflex in rats. Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically-induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT-PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels.

Patterns of Phrenic Nerve Discharge after Complete High Cervical Spinal Cord Injury in the Decerebrate Rat.

Studies conducted since the second half of the 19th century have revealed spontaneous as well as pharmacologically induced phasic/rhythmic discharge in spinal respiratory motor outputs of cats, dogs, rabbits, and neonatal rats following high cervical transection (Tx). The extent to which these various studies validate the existence of a true spinal respiratory rhythm generator remains debated. In this set of studies, we seek to characterize patterns of spontaneous phasic/rhythmic, asphyxia-induced, and pharmacologically induced activity occurring in phrenic nerve (PhN) discharge after complete high cervical (C1-C2) spinal cord transection. Experiments were performed on 20 unanesthetized decerebrate Sprague-Dawley adult male rats. Patterns of spontaneous activity after spinalization included tonic, phasic, slow oscillatory, and long-lasting tonic discharges. Topical application of antagonists of GABAA and glycine receptors to C1- and C2- spinal segments induced left-right synchronized phasic decrementing activity in PhN discharge that was abolished by an additional C2Tx. Asphyxia elicited increases in tonic activity and left-right synchronized gasp-like bursts in PhN discharge, demonstrating the presence of spinal circuits that may underlie a spinal gasping-like mechanism. We conclude that intrinsic slow oscillators and a phasic burst/rhythm generator exist in the spinal cord of the adult rat. If present in humans, this mechanism may be exploited to recover respiratory function in patients sustaining severe spinal cord injury.