AJP-Regu Articles in PresS. Published on July 11, 2002 as DOI 10.1152/ajpregu.00075.2002
IN VIVO ELECTROPHYSIOLOGICAL RESPONSES OF PEDUNCULOPONTINE NEURONS TO STATIC MUSCLE CONTRACTION
Edward D. Plowey1, Jeffery M. Kramer1, Joseph A. Beatty1 and Tony G. Waldrop2*
1
Department of Molecular and Integrative Physiology University of Illinois at Urbana-Champaign Urbana, IL, USA 2
Department of Cell and Molecular Physiology University of North Carolina at Chapel Hill Chapel Hill, NC, USA
Running Head: Pedunculopontine Responses to Muscle Contraction Number of Pages: 31 pages of text, 8 figures and 3 tables *Address for Correspondence: Tony G. Waldrop Department of Cell and Molecular Physiology 312 South Building, CB # 400 Chapel Hill, NC 27599 Voice: 919-962-1319 Fax: 919-962-1476 e-mail:
[email protected] URL: www.med.unc.edu/wrkunits/2depts/physiolo/pages/waldrop.htm
Copyright 2002 by the American Physiological Society.
2
Abstract The pedunculopontine nucleus (PPN) has previously been implicated in central command regulation of the cardiorespiratory adjustments that accompany exercise. The current study was executed to begin to address the potential role of the PPN in the regulation of cardiorespiratory adjustments evoked by muscle contraction. Extracellular single-unit recording was employed to document the responses of PPN neurons during static muscle contraction. Sixty-four percent (20/31) of neurons sampled from the PPN responded to static muscle contraction with increases in firing rate. Furthermore, muscle contraction responsive neurons in the PPN were unresponsive to brief periods of hypotension, but were markedly activated during chemical disinhibition of the caudal hypothalamus (CH). A separate sample of PPN neurons was found to be moderately activated during systemic hypoxia. Chemical disinhibition of the PPN was found to markedly increase respiratory drive. These findings suggest that the PPN may be involved in modulating respiratory adjustments that accompany muscle contraction and that PPN neurons may have the capacity to synthesize muscle reflex and central command influences.
Keywords pedunculopontine nucleus; muscle contraction; respiration
3
Introduction Central command and muscle reflex mechanisms are thought to be involved in driving cardiorespiratory adjustments observed during the onset of moderate exercise (32, 41, 43). The central command hypothesis states that specific brain areas are responsible for parallel, feed-forward activation of brainstem locomotor and cardiorespiratory loci during exercise (32, 43). Excitation of cardiorespiratory centers is also driven by muscle reflex pathways that are stimulated by the mechanical and metabolic products of active muscles during exercise (26). The capacity of specific brain areas to potentially contribute to both central command and muscle reflex influences has been documented in the caudal hypothalamus (44), the ventrolateral medulla (33) and the dorsal horn of the spinal cord (9). Several authors have hypothesized that orchestration of influences from central command and muscle reflex pathways partly underlies the ability of the central nervous system to evoke alterations in cardiorespiratory drive that are appropriately matched to the metabolic demand of physical activity (32, 33, 35, 43, 44). The pedunculopontine nucleus (PPN) has garnered attention as a potential regulator of cardiorespiratory drive during exercise as a component of the mesencephalic locomotor region (MLR) (11, 43). The MLR of the rat, located in the mesencephalic tegmentum at the lateral extent of the brachium conjunctivum, is an area from which coordinated locomotion can be evoked via electrical stimulation or chemical disinhibition in a non-anesthetized, decerebrate preparation (16). Garcia-Rill and colleagues demonstrated that the MLR of the rat is highly coexistent with the cholinergic, NADPH-diaphorase positive neurons of the PPN (17, 18). The PPN is known to be active during locomotion, as has been shown by extracellular neuronal
4
recordings in cats (19) and via examination of c-fos expression in the PPN following treadmill exercise in rats (25). Activation of the MLR in cats produces feed-forward increases in efferent cardiorespiratory drive that parallel concurrent increases in locomotor drive, yet persist in the absence of elevated muscle feedback during fictive locomotion (11). The capacity of the MLR to produce feed-forward increases in cardiovascular drive has been documented in rats as well (4, 8). Given the apparent importance of the PPN as an anatomical component of the MLR (17, 18), it is reasonable to hypothesize that the PPN may play a role in the regulation of the cardiorespiratory adjustments that accompany exercise though a central command mechanism. To begin to evaluate the possibility that the PPN contributes a potential modulatory influence to the cardiorespiratory responses evoked by muscle contraction, we determined if neurons of the PPN and the surrounding mesencephalic tegmentum respond to evoked static contraction of the hindlimb muscles in anesthetized rats using single-unit extracellular recording. We hypothesized that if the PPN modulates the cardiorespiratory adjustments evoked by muscle contraction, then neurons sampled from the PPN will exhibit alterations in firing rate during static muscle contraction. The data presented suggest that the firing rates of PPN neurons are enhanced during evoked muscle contraction in anesthetized rats and that activation of the PPN may, as observed during muscle contraction, have an impact on respiratory drive.
Methods All of the procedures described in this paper were executed under animal experimentation protocols that were approved by the Laboratory Animal Care Advisory
5
Committee of the University of Illinois at Urbana-Champaign. These procedures are in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Animal Preparation Male Sprague-Dawley rats (220-350 grams, 63 total animals) were anesthetized with intraperitoneal injections of a mixture of α-chloralose (65 mg/kg) and urethane (800 mg/kg) dissolved in Ringer’s. Adequate depth of anesthesia was maintained via anesthetic supplements that were administered intravenously upon evidence of a positive foot withdrawal response to noxious pinch or of a positive eyeblink response to tactile stimulation of the cornea. The trachea was cannulated with PE-205 tubing (Clay Adams, Parsippany, NJ) to maintain a patent upper respiratory tract and facilitate spontaneous ventilation of 100% O2. Catheters (PE-50 tubing; Clay Adams) filled with heparinized saline (75µg/ml heparin; Sigma, St. Louis, MO) were inserted into the left external jugular vein and left common carotid artery to allow drug administration and measurement of cardiovascular variables, respectively. Pulsatile arterial blood pressure was monitored via a Model P23 pressure transducer (Gould, Inc., Oxnard, CA) connected to the arterial catheter. Heart rate was derived from the voltage output of the pressure transducer using a biotachometer (Gould). Rats were then placed prone in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) to immobilize the cranium. A rectal temperature probe, a radiant heat lamp and a water perfused heating pad were employed to maintain the animal’s body temperature at 37 + 1°C. The bite bar of the stereotax was then adjusted to the vertical
6
level at which the lambda and bregma skull coordinates were measured in the same horizontal plane. A parietal craniotomy was performed over the mesencephalon in all animals and was extended over the diencephalon in some subjects. Differential tefloncoated stainless steel wire (0.0055 inches in diameter; A-M Systems, Inc., Carlsborg, WA) electrodes were inserted into the diaphragm using a 23-guage hypodermic needle. The electrical activity measured by the diaphragmatic electrodes was amplified (3003000 Hz bandwidth; P5 Series AC Pre-amplifier, Grass Instruments, Quincy, MA) full wave rectified and integrated (Gould Integrator Amplifier) to yield an electrical correlate of respiratory activity known as integrated diaphragmatic electromyogram activity, or ³DEMG. The ³DEMG signal was sent to a biotachometer (Gould) to derive the respiratory rate (f) of the animal. The product of f and the average of the peak ³DEMG amplitude, known as minute ³DEMG amplitude, was examined as an electrophysiological indicator of relative changes in the minute ventilation. In 37 animals prepared for muscle contraction experiments, right hindlimb muscle contraction was evoked by electrical stimulation of the right tibial nerve. The tibial nerve was accessed through an incision of the skin of the posterior thigh. The muscles of the posterior compartment were bluntly dissected to expose the origins of the tibial, sural and peroneal branches of the sciatic nerve. The tibial nerve was dissected from the sural and peroneal nerves and placed on a shielded bipolar platinum electrode. The nerve was covered in a pool of warm mineral oil to prevent dessication. Limb movement was prevented by placement of a precision clamp about the knee. To evoke static contraction of the hindlimb muscles, the tibial nerve was electrically stimulated (40 Hz, 1 msec square wave pulses) at 2X motor threshold (MT) for 30 seconds.
7
Data Collection Single unit extracellular recordings were made with high impedance electrodes (3-6 MΩ; FHC, Inc., Bowdoinham, ME) stereotaxically placed into the PPN. Recording tracts were executed within the following coordinates according to Paxinos and Watson (34): 0.3 to 1.7 mm rostral, 1.4 to 2.2 mm lateral, and 5.5 to 1.0 mm dorsal to interaural zero. Extracellular activity was amplified (100k; P5 Series AC Pre-amplifier, Grass) and filtered (300-1000 Hz band width). Single units were isolated with a window discriminator (FHC, Inc.). Action potentials that fell within the recording window triggered a TTL pulse that were sent to a rate meter (FHC) and to a digital chart recorder (Windows based PC running PowerLab v.3.4.4, AD Instruments, Inc., Grand Junction, CO). Discriminated action potentials were also sent to a storage oscilloscope to check for consistency of the action potential signature to thus ensure a stable recording of a single unit. Extracellular activity was also sent to a speaker to monitor unit activity audibly. Recording tracts were performed in both the right (ipsilateral) and left (contralateral) sides of the mesencephalon. The responses of isolated mesencephalic units were recorded during 30 seconds of static contraction of the hindlimb muscles. Following a 15 minute recovery period, response reliability was tested during a subsequent period of muscle contraction. To evaluate the possibility that neuronal responses observed during muscle contraction were evoked by alterations in arterial pressure, the responses of the neurons to brief periods of hypotension or hypertension induced by intravenous injections of sodium nitroprusside (SNP; 5-10µg; Sigma) or phenylephrine (PHE; 3-5µg; Sigma), respectively, were documented. To test the
8
possibility that we were directly activating afferents via electrical stimulation to evoke responses in PPN neurons, five muscle contraction-responsive neurons were recorded during electrical stimulation of the tibial nerve at 2X MT after the nerve was crushed just distal to the stimulation electrode. In all experiments, we made sure that stimulation of the crushed tibial nerve at 2X MT failed to evoke cardiorespiratory adjustments through direct activation of tibial nerve afferents. For a subset of 6 PPN neurons that responded to muscle contraction, the responses to supramesencephalic activation of central command were recorded. Feedforward increases in cardiorespiratory drive, in the absence of locomotion, were evoked via microinjections of the GABAA receptor antagonist bicuculline (BIC) (5mM in Ringer’s, 60nl; Sigma) into the caudal hypothalamus (CH) (11, 42). A microinjection pipette (2030 micron tip aperture) was pulled from a glass capillary tube (1mm diameter; World Precision Instruments, Inc., Sarasota, FL) with a one-stage, upright pipette puller (Narishige, Tokyo, Japan). The pipette was stereotaxically placed in the CH using the following coordinates: rostral +4.8mm; lateral +0.5mm; dorsal +1.7mm relative to interaural zero (34). Microinjections were made with a PV800 Pneumatic PicoPump (World Precision Instruments, Inc.) and were measured by monitoring the movement of the meniscus of the injectate through a calibrated microscope reticule (Reichert Scientific Instruments, Buffalo, NY). The firing behavior of PPN neurons were recorded for at least two minutes prior to the microinjection of bicuculline, throughout the period of CH activation, and for at least two minutes following the return of the cardiorespiratory responses to baseline. In separate experiments (9 rats), similar animal preparations, except for preparation of the hindlimb for muscle contraction, were used to document the
9
responses of PPN neurons to systemic hypoxia. Hypoxia was induced for one minute periods by switching the inspired gas from 100% O2 to a gas mixture composed of 10% O2/90% N2. The effects of the hypotension observed during hypoxia on neuronal firing rate were evaluated via intravenous injections of SNP as described above. The seventeen remaining subjects were prepared similarly in order to chemically activate the PPN. Single-barrel microinjection pipettes filled from the tip via suction with BIC (5mM in Ringer’s, 60nl; Sigma), vehicle and 0.5% Chicago sky blue dye (Sigma) in the opposite order of injection. Solutions were separated via thin layers of mineral oil. The microinjection pipettes were stereotaxically placed in the PPN or surrounding mesencephalic tegmentum using the coordinates employed for extracellular recording. Stable baseline cardiorespiratory variables were recorded for 5 minutes prior to injection of BIC into the PPN or control sites, and then for the duration of observed cardiorespiratory responses. Cardiorespiratory variables were also observed after injection of vehicle and/or Chicago sky blue to ensure that responses to bicuculline were specific.
Histology Following the termination of successful recording tracts or injection experiments in the PPN, animals were prepared for histological analyses of recording and injection sites. The positions of recorded neurons were demarcated with DC electrolytic lesions (300 µA, 8-10 seconds). Injection sites in the PPN and CH were marked with 60nl microinjections of Chicago sky blue dye following the experiments. Animals were then deeply anesthetized with a supplemental injection of α-chloralose/urethane (1/4 initial pre-surgical dose) and perfused transcardially with heparinized saline followed by 4%
10
paraformaldehyde in phosphate buffered saline/1mM MgCl2. The brain was post-fixed in the fixative for 2 hours and then infiltrated with 20% sucrose/5mM MgCl2. Midbrains containing the PPN were sliced on a sliding microtome (American Optical Company, Buffalo, NY) with a freezing stage (Sensortek, Clifton, NJ) into 30 micron sections. Alternate sections were either mounted on gelatin-coated slides and stained with neutral red or were incubated in 1mM NADP+, 0.2mM nitroblue tetrazolium, 15mM sodium malate in 0.1M Tris buffer for 45 minutes to reveal the NADPH-diaphorase positive neurons of the pedunculopontine nuclei (39). Diencephalons containing the caudal hypothalamus were sectioned into 50 micron slices and mounted on gelatin coated slides. Alternate sections were either stained with neutral red (Sigma) or left unstained to allow determination of the position of the microinjection site.
Data Analyses Cardiorespiratory and electrophysiological variables were recorded to the Powerlab digital chart recorder and subsequently analyzed. Cardiorespiratory and electrophysiological responses to muscle contraction were determined by contrasting the means of the variables during the one minute period immediately prior to muscle contraction (baseline) with the means of the variables during the entire thirty second period of muscle contraction. Baseline variables were also contrasted to the peak responses observed during muscle contraction. In addition, the standard deviations of the firing rates from the mean over the entire baseline period were determined. Individual neurons were labeled as responsive (increase or decrease) if the difference between the mean firing rate before muscle contraction and the mean firing rate during muscle contraction exceeded one standard deviation. The means of the
11
cardiorespiratory and electrophysiological variables before and during muscle contraction were contrasted using two-tailed, paired Student’s t-tests, with p0.05). In addition, consistent with the lack of responses to SNP-evoked changes in blood pressure, three PPN neurons in this sample did not exhibit alterations in firing rate when blood pressure was transiently elevated via intravenous injection of phenylephrine. We were also interested in testing whether the cardiorespiratory and neuronal responses we observed were due to activation of muscle afferents by muscle contraction or due to direct activation of sensory afferents in the tibial nerve by our stimulation electrode. In all experiments, stimulation of the tibial nerve at 2X MT after the nerve was crushed distal to the stimulation electrode failed to evoke changes in arterial pressure, heart rate, respiration and muscle tension. In addition, five muscle reflex responsive PPN neurons were recorded during electrical stimulation of the tibial nerve after the nerve was crushed distal to the stimulation site (Fig. 5). In all five cases, stimulation of the crushed tibial nerve at 2X MT failed to reproduce the neuronal responses observed during muscle contraction, while stimulation at higher voltages did result in activation of PPN neurons.
14
The responses of 13 PPN neurons during systemic hypoxia, induced by spontaneous ventilation of 10% O2, were documented in separate animals. Figure 6 depicts the response of one PPN unit during one minute of exposure to the hypoxic gas mixture. Note that the neuron exhibited an increase in firing rate that coincided with the decrease in arterial pressure and increases in heart rate and respiration. Eight of the 13 PPN units tested responded with increases in firing rate, two responded with decreases in firing rate, and three failed to respond during one minute of systemic hypoxia. The basal firing rate of this sample of PPN units was 7.9 + 1.2 Hz. The firing rate of the entire sample increased to 12.4 + 1.9 Hz (p
