Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 93: 1448-1456, 2002. First published June 14, 2002; doi:10.1152/japplphysiol.00225.2002
8750-7587/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
93/4/1448    most recent
00225.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fenik, V.
Right arrow Articles by Kubin, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fenik, V.
Right arrow Articles by Kubin, L.
Vol. 93, Issue 4, 1448-1456, October 2002

A5 cells are silenced when REM sleep-like signs are elicited by pontine carbachol

Victor Fenik, Vitaliy Marchenko, Patrick Janssen, Richard O. Davies, and Leszek Kubin

Department of Animal Biology, School of Veterinary Medicine, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The A5 noradrenergic neurons are considered important for cardiorespiratory regulation. We hypothesized that A5 cells are silenced during rapid eye movement (REM) sleep, thereby contributing to cardiorespiratory changes and suppression of hypoglossal (XII) motoneuronal activity. We used an anesthetized, paralyzed, and artificially ventilated rat in which pontine microinjections of carbachol trigger signs of REM sleep, including hippocampal theta rhythm, motor suppression, and silencing of locus coeruleus neurons. All 16 putative noradrenergic A5 cells recorded were strongly suppressed when the REM sleep-like episodes were elicited and also after intravenous clonidine. Antidromic mapping showed that none of six neurons tested projected to the XII nucleus, whereas three of five projected to the nucleus of the solitary tract and two of four to the rostral ventrolateral medulla. Bilateral microinjections of clonidine into the A5 regions did not alter XII nerve activity. These data suggest that A5 neurons are silenced during natural REM sleep. This will lead to decreased norepinephrine release and may alter synaptic transmission in the nucleus of the solitary tract and rostral ventrolateral medulla without, however, a detectable impact on XII motoneurons.

hypoglossal motoneurons; norepinephrine; nucleus of the solitary tract; pons; rapid eye movement sleep


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NOREPINEPHRINE-CONTAINING neurons of the A5 group, located in the ventrolateral pons between the root of the facial nerve and the superior olive, are considered important regulators of cardiorespiratory function (reviewed in Refs. 11, 24, 25, 45, 46). They have extensive axonal projections that include cardiorespiratory and motor regions of the brain stem and spinal cord (1, 8, 9, 23, 36). Through these projections, they may control, among others, sympathetic and respiratory outputs and, via projection to the hypoglossal (XII) motor nucleus, may mediate noradrenergic excitation of XII motoneurons (5, 22, 27, 40, 49). XII motoneurons are of particular interest because they innervate the genioglossus muscle of the tongue, an important airway dilator. Its decreased activity during sleep contributes, in predisposed individuals, to the pathophysiology of the obstructive sleep apnea syndrome (34).

The noradrenergic neurons of the locus coeruleus (LC) and the sub-LC region show a robust relationship of their firing frequency with the sleep-wake cycle: the highest activity occurs during wakefulness, and the lowest during the rapid eye movement (REM) stage of sleep (6, 43). Consistent with these findings, the extracellular level of norepinephrine is reduced in the XII nucleus during the motor atonia produced by electrical stimulation of the pontine reticular formation region implicated in the triggering of REM sleep (35). The noradrenergic cells of the A5 group may be similarly modulated with the sleep-wake cycle, but this has not been tested. Their reduced or abolished activity during sleep would contribute to the withdrawal of the effects that these neurons exert on their postsynaptic targets, including their putative excitatory postsynaptic effects on XII motoneurons.

To gain insight into changes in A5 cell activity that may occur during REM sleep, we used a recent variant of many carbachol models of REM sleep that have been developed since pontine cholinergic mechanisms were identified as crucial for generation of this state (reviewed in Refs. 7, 33). In the model that we used, carbachol, a cholinergic agonist, is microinjected into the dorsomedial pons of urethane-anesthetized, paralyzed, vagotomized, and artificially ventilated rats to produce REM sleep-like signs comprising cortical electroencephalogram (EEG) activation, hippocampal theta rhythm, suppression of XII nerve activity, and silencing of noradrenergic cells of the LC. This approach offers stable experimental conditions, whereas the XII nerve and two forebrain signals (cortical and hippocampal) provide the outputs by which one can assess the similarity of the carbachol-elicited condition to REM sleep. The REM sleep-like episodes can be elicited repeatedly, thus offering a tool with which one can characterize changes in neuronal activity and use invasive techniques to map neuronal projections.

Because A5 neurons have widespread projections, we focused our study on those A5 cells that project toward the dorsomedial medulla, including the XII nucleus, and to the rostral ventrolateral medulla (RVLM), another target of A5 neurons important for cardiorespiratory control (11, 24, 25, 45). We used the antidromic mapping technique to identify axonal projections of A5 neurons and assessed changes in their activity at the time when carbachol elicited REM sleep-like signs. Preliminary reports were published (19, 20).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation and recording procedures. Experiments were performed on 24 adult Sprague-Dawley rats [body weight: 395 ± 48 (SD) g]. The procedures for anesthesia, surgery, and recording followed the guidelines of the Institute for Laboratory Animal Research and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

The animals were anesthetized with isofluorane (2%) followed by urethane (1 g/kg ip, supplemented by 50-mg iv injections as needed). The trachea was exposed and intubated, and a femoral artery and vein were cannulated for arterial blood pressure monitoring and fluid injections, respectively. One XII nerve was cut peripherally, freed from the surrounding tissue, and placed in a cuff-type recording electrode (18). Both vagi were cut in the neck to enhance XII nerve activity and make it independent of lung volume feedback. The animal was placed in a stereotaxic head holder; two openings were drilled in the caudal, medial aspect of the right parietal bone; and the dura was removed for insertion of a carbachol-containing pipette and hippocampal recording electrode. Another opening was made in the interparietal bone for inserting a recording or microinjection pipette into the A5 region. The caudal medulla was exposed by a posterior fossa craniotomy to insert a stimulating electrode. For monitoring the cortical EEG, two screws were attached to the scull: one in the frontal bone 2 mm anterior and 2 mm to the right and one into the parietal bone 3 mm posterior and 2 mm to the left of the bregma. To record hippocampal activity, two insulated wires with tips separated by 0.8 mm were placed in the dorsal hippocampus, 3.7 mm posterior to and 2.2 mm to the right of the bregma and 2.4 mm below the cortical surface. The position of this electrode was adjusted to maximize the amplitude of the theta rhythm elicited by a strong foot pinch.

The animals were paralyzed (pancuronium bromide, 2 mg/kg iv, supplemented with 1 mg/kg injections as needed) and artificially ventilated with an air-oxygen mixture (final O2 concentration 30-60%) at 50-70 lung inflations/min. Intravenous fluids (124 mM NaCl, 30 mM NaHCO3, 2% glucose) were continuously infused at a rate of 3 ml · kg-1 · h-1. After paralysis, the level of anesthesia was assessed by continuously monitoring the arterial blood pressure and XII nerve activity and intermittently applying a pinch to the tail or hindlimb. A regular respiratory rhythm, steady XII nerve electroneurogram, and blood pressure, and only transient changes in cortical and hippocampal signals, similar to those before paralysis, indicated that the animal was adequately anesthetized. The rectal temperature and end-expiratory CO2 (Columbus Instruments capnograph) were continuously monitored. All recordings were obtained from animals with a systolic arterial blood pressure >85 mmHg, rectal temperature of 36-37°C, and end-expiratory CO2 adjusted to maintain a steady respiratory modulation of XII nerve activity (5-6%).

Electrophysiological recordings and stimulation. Extracellular single-cell activity was recorded with glass pipettes having tip diameters of 2.5-3.0 µm and filled with 0.5 M Na acetate and 2% Pontamine blue, which had a resistance of 4-5 MOmega at 1 kHz. The pipettes were held in a hydraulic manipulator (Haer) and inserted at an angle of 7° lateral to avoid penetration of the lateral sinus. The recording sites were marked by the iontophoretic deposition of Pontamine blue dye (5 µA, 10 min) (Fig. 1).


View larger version (61K):
[in this window]
[in a new window]
  		Fig. 1.   The anatomic localization of noradrenergic A5 cell recording sites and pontine sites from which carbachol elicited rapid eye movement (REM) sleep-like episodes. A: the site of recording from 1 of the studied A5 cells (arrow) shown in a brain section immunohistochemically stained for tyrosine hydroxylase. The site is located near a cluster of noradrenergic cells. B: distribution of 15 sites at which A5 cells were recorded in this study. C: distribution of 15 sites at which carbachol injections (10 nl) elicited REM sleep-like episodes while we simultaneously recorded from individual A5 neurons. The sites in B and C are superimposed on standard brain sections taken from Paxinos and Watson (41) at the indicated anteroposterior levels from bregma. 7n, Facial nerve; LSO, lateral superior olive; PO, nucleus pontis oralis; PPT, pedunculopontine tegmental nucleus; py, pyramidal tract; scp, superior cerebellar peduncle; VII, facial nucleus; VT, ventral tegmental nucleus.

Cortical, hippocampal, A5 cell, and XII nerve activity were recorded with alternating-current amplifiers (N101, Neurolog or P-5, Grass) with bandwidths set at 1-100 and 3-8 Hz for the cortical and hippocampal signals, respectively, and 30-2,500 Hz for the remaining signals. To obtain a smooth record of changes in activity with the central respiratory rhythm, XII nerve activity was full-wave rectified and passed through a moving-average circuit (time constant 100 ms; MA-821 RSP; CWE). These signals, together with an event marker, arterial blood pressure, tracheal pressure, and end-expiratory CO2, were monitored on a chart recorder (TA-11; Gould Instruments) and recorded on a 16-channel digital tape recorder (C-DAT; Cygnus Technology).

Antidromic mapping was performed by using platinum-plated tungsten microelectrodes (0.8-1.2 MOmega ; FHC). The electrodes were inserted in the medulla to stimulate either the RVLM region at a depth of 2.0-3.5 mm or the dorsomedial medulla at a depth of 0.3-2.5 mm. Stimulation was performed by using single pulses of negative current having an amplitude of 1-500 µA and a duration of 0.5 ms (Grass S88). Antidromic activation was verified by using the collision test. For each antidromically activated cell, the depth-threshold relationship was obtained by moving the stimulating electrode in 50-µm steps. In each experiment, one stimulation site was marked with an electrolytic lesion (20 µA, 40 s).

Carbachol and clonidine microinjections. For carbachol injections, a glass pipette (A-M Systems, tip diameter 20-30 µm) filled with 10 mM carbachol (carbamylcholine, Sigma Chemical) and 2% Pontamine in 0.9% saline was inserted into the dorsomedial pontine reticular formation by using surface landmarks established in earlier studies (50). Ten nanoliters of carbachol were injected over 10-20 s by applying pressure to the fluid in the pipette while monitoring the movement of the meniscus with a calibrated microscope. Only the effects of those injections that caused the characteristic REM sleep-like changes in the EEG and hippocampal signals (see RESULTS) were analyzed.

In some experiments, clonidine (RBI), an alpha 2-adrenoceptor agonist, was injected bilaterally into the A5 region (0.75 mM of clonidine and 2% Pontamine in 0.9% saline, 20-nl injections). The pipettes of the same type and size as for carbachol were positioned in a manner similar to the recording electrode for A5 neurons. Only the effects of injections whose locations were confirmed histologically were included in the analysis.

Experimental protocol and data analysis. The recorded cells were identified as putative noradrenergic A5 neurons when they fulfilled the following criteria (26, 32, 39, 42): 1) they had a low and steady firing rate (<5 Hz) and long-duration (>0.6 ms) action potentials; 2) their activity was inhibited by clonidine (2-4 µg iv); and 3) they were recorded in sites having coordinates corresponding to the location of the A5 group and subsequently found in the A5 region. In some cases, the presence of noradrenergic neurons at the recording site was additionally verified by tyrosine hydroxylase immunohistochemistry.

We searched with the recording electrode for cells in the A5 region while applying stimulating pulses (0.5 Hz) in either the RVLM or XII nucleus. Once a stable recording from a spontaneously active, putative A5 cell was achieved, with or without a response to antidromic activation, tests were performed in the following order: microinjection of carbachol into the pons, antidromic mapping of the cell's projections, and an intravenous injection of clonidine.

For the effective carbachol injections, 30-s segments of records centered around the point of maximal response were analyzed. The measurements taken during these periods were compared with those obtained from a 30-s period preceding carbachol injections. The amplitude of XII nerve activity was measured from the moving average of the signal between the level in expiration, when there was usually no activity, and the peak activity in inspiration. Carbachol-induced changes were expressed relative to the amplitude of the respiratory modulation of XII nerve activity during the precarbachol period. The central respiratory rate was determined from the record of XII nerve activity. The durations of the carbachol responses were measured based on changes in hippocampal activity, which typically have a clear onset and offset (33). The latencies of the responses were assessed from the onset of the injection to the start of the response.

In experiments with clonidine injections into the A5 region, measurements of XII nerve activity, respiratory rate, and blood pressure were performed at 30-s intervals, starting just before the first injection (baseline) and continuing past the second of the bilateral injections.

Histology and immunohistochemistry. At the conclusion of the experiment, an overdose of urethane was injected (2 g/kg iv), one jugular vein was exposed and cut, and the animal was perfused intra-arterially with 50 ml of saline followed by 100 ml of phosphate-buffered 10% formalin. For identification of the stimulation and carbachol or clonidine injection sites, parasagittal or coronal 100-µm sections were cut on a vibratome, mounted, and counterstained with Neutral red. For identification of the recording sites, tissue blocks were cryoprotected in 30% sucrose, and 35-µm coronal or parasagittal sections were cut on a cryostat. Each recording, stimulation, and carbachol or clonidine injection site was placed on a corresponding standard cross section taken from a rat brain atlas (41).

For immunohistochemical verification that cell recordings were obtained from regions containing noradrenergic neurons, free-floating sections containing the recording site were incubated for 48-63 h at 4°C in phosphate-buffered saline containing anti-tyrosine hydroxylase antibodies (1:40,000; catalog number T-1299, Sigma Chemical), 0.2% Triton X, and 5% horse serum. Subsequently, they were successively incubated for 1 h at room temperature in biotinylated anti-mouse antibodies and avidin-biotin-horseradish peroxidase complex (ABC kit, Vector Laboratories). Horseradish peroxidase was then visualized by using diaminobenzidine and nickel ammonium sulfate, as previously described (16). Sections were mounted, counterstained with Neutral red, and coverslipped.

Statistical analysis. Statistical analysis was performed by using SigmaStat (Jandel). ANOVA and paired or unpaired, two-tailed Student's t-tests were used to compare the measurements obtained after different treatments and the properties of different groups of cells. When t-tests were used, it was first verified that the data were normally distributed. The variability of the data is characterized by the SD. Differences were considered significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Behavior of A5 neurons during carbachol-induced REM sleep-like episodes. Because our goal was to assess the changes in the discharge of putative A5 cells during carbachol-induced REM sleep-like episodes, this report includes only the results from cells that were tested with carbachol and fulfilled our criteria for A5 noradrenergic neurons. Sixteen such cells were recorded in 16 rats. Their action potentials had a duration of 0.75 ± 0.1 ms and were followed by a slow afterpotential of opposite polarity (2.9 ± 1.5 ms). The mean control firing rate of these cells was 1.6 ± 1.0 Hz (range: 0.26-3.6 Hz). For eight cells, the brain sections containing the marked recording site were subjected to tyrosine hydroxylase immunohistochemistry. In all cases, noradrenergic neurons were found in close vicinity to the recording site (Fig. 1A). Fifteen of the 16 recording sites were recovered histologically; all were localized to a region dorsal, dorsolateral, and ventrolateral to the superior olive, near the roots of the facial nerve (Fig. 1B).

Each of the 16 cells was recorded during a pontine injection of carbachol that produced characteristic REM sleep-like changes in the cortical and hippocampal EEG and a profound suppression of XII nerve activity (33, 50). Figure 1C shows the location of 15 carbachol injection sites (one site was not recovered). The latency of the REM sleep-like episodes following the onset of the injection was 47 ± 44 s (range: 7-155 s), and their average duration was 3.1 ± 0.8 min (range: 1.6-4.7 min) (n = 16). In 14 out of the 16 cases, the baseline level of XII nerve activity was sufficiently high to assess its changes after carbachol. At the beginning of the carbachol-induced episodes, both XII nerve activity and respiratory rate decreased. The average maximal suppression of XII nerve activity was to 8.2 ± 15% (range: 0-41%) of control (P < 0.001; n = 14). In nine cases, the activity in the XII nerve was abolished after carbachol for 124 ± 57 s, making it impossible to determine the maximal slowing of the respiratory rate. When XII nerve activity was preserved throughout the episodes, the respiratory rate decreased from 50 ± 7 to 32 ± 13 breaths/min (P < 0.05; n = 5), or by 36 ± 23% of control, and the maximal suppression of XII nerve activity was to 36 ± 21% (range: 12-67%) of control.

Thirteen of the 16 cells were silenced during the REM sleep-like episodes. One example is shown in Fig. 2A. The average cell firing rate decreased from 1.6 ± 1.0 to 0.1 ± 0.1 Hz and recovered to 1.3 ± 0.9 Hz after the episodes (n = 16). The average duration of the silent period was 143 ± 63 s (range: 41-265 s). The activity of the three cells that were not silenced was depressed from 2.9 ± 0.9 to 0.3 ± 0.07 Hz (P < 0.05). The dependence of the level of the cell's activity on the three successive conditions of the protocol (baseline, carbachol, and recovery) was highly significant when tested with one-way repeated-measures ANOVA (F2,15 = 37, P < 0.001). The post hoc pairwise comparisons with Bonferroni's correction revealed significant differences between the baseline level of activity and carbachol, and between carbachol and recovery, whereas the difference between baseline and recovery was not significant.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2.   Recording from a putative noradrenergic A5 cell during the REM sleep-like episode produced by pontine carbachol (A) and during the systemic administration of clonidine (B). A: the occurrence of the REM sleep-like response is marked by the simultaneous appearance of theta rhythm in the hippocampus (top trace, with 5-s insets showing portions of the trace at an expanded time scale) and a profound suppression of hypoglossal (XII) nerve activity (bottom trace, showing the moving average of the raw signal). The cell is reversibly silenced during both tests. EEG, electroencephalogram.

Fourteen of the 16 cells were tested with an intravenous injection of clonidine. The activities of 13 cells were abolished (Fig. 2B). The activity of one remaining cell (silenced by carbachol) was depressed by clonidine (4 µg iv) from 2.4 to 0.5 Hz. Figure 3 shows the firing rates of all cells tested, measured at successive stages of the protocol with carbachol and clonidine.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 3.   Firing rates of 16 putative noradrenergic A5 cells at successive stages of the protocol that included eliciting the REM sleep-like episode by pontine microinjection of carbachol (10 nl, 10 mM), recovery from the effect of carbachol, and systemic administration of the alpha 2-adrenergic agonist clonidine (2-4 µg iv). Each cell is represented by a unique symbol. All cells were silenced, or nearly silenced, during the REM sleep-like episodes, after which their activity promptly recovered and was then profoundly suppressed or abolished by clonidine.

Axonal projections of A5 neurons. The axonal projections of 10 A5 cells were investigated. Figure 4 shows examples of antidromic responses and collision tests for one cell whose two distinct axonal branches were stimulated in the dorsomedial medulla. Stimulation of one branch evoked an action potential with a latency of 26 ms (Fig. 4A) and the other with a latency of 16 ms (Fig. 4C).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Antidromic activation of an A5 cell from the nucleus of the solitary tract (NTS). Two distinct antidromic latencies were observed at different stimulus intensities. A and B: collision test for the longer latency (26 ms, stimulus 55 µA). B: the delay between the spontaneous action potential occurring at the beginning of the record and the stimulus () was ~2 ms shorter than in A. The evoked response did not occur due to collision. C and D: collision test for the shorter latency (16 ms, stimulus 150 µA) shown in the same format as in A and B. E: the ability of the same cell to respond to 2 stimuli applied at an interval shorter than the shortest interval between the spontaneous and evoked action potential elicited during the collision tests (stimulus 150 µA) shows that the absence of evoked action potentials in B and D was not due to refractoriness. Each record contains 3 superimposed traces.

Most of the penetrations with the stimulating microelectrode were made within the dorsomedial medulla, aiming at the XII nucleus. The tracks coursed through the nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus, XII nucleus, and the dorsomedial reticular formation ventral to the XII nucleus, as shown in Fig. 5A. We carefully searched for the projections of six A5 cells in the XII nucleus but found none. In contrast, three of the five cells whose axons were adequately searched for within both the NTS and XII nucleus had minimum threshold points located only in the NTS. Each of these three cells had several axonal branches, with each having a distinct antidromic latency and distinct location (depth) of the minimum threshold point (Fig. 5B). A fourth cell projected to the reticular formation ventral to the XII nucleus (its projections to the NTS were not tested). For only two cells, no projections were found in the dorsomedial medulla. Thus none of the six cells tested had axonal ramifications in the XII nucleus (Fig. 5C).


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 5.   Localization of minimum threshold points for antidromic activation of A5 cells from medullary cardiorespiratory regions. A: microphotograph of a parasagittal medullary section (0.3 mm lateral from midline) containing an electrolytic lesion (arrow) made in the NTS at 1 of the minimum threshold points for antidromic activation of an A5 cell. The line shows the direction of penetration with the stimulating microelectrode. B: depth-threshold curves obtained from the track in A. To determine the position of the minimum threshold sites, experimental points () were extrapolated by second-order polynomials (see Ref. 12). C and D: localization of all minimum threshold points (; n = 13) identified by antidromic activation of 6 A5 cells shown on 2 parasagittal medullary sections (0.3 mm and 2.2 mm lateral from midline). AP, area postrema; DMV, dorsal motor nucleus of the vagus; IO, inferior olive; LRN, lateral reticular nucleus; NA, nucleus ambiguus.

For the three cells with several axonal branches, the shortest antidromic latencies averaged 12 ± 5 ms (range: 7-16 ms), and the longest were 20 ± 7 ms (range: 13-26 ms). The average difference between the shortest and the longest latency was 8.7 ± 7 ms (range: 1-15 ms). The average minimum threshold for all branches was 78 ± 71 µA (range: 4.8-190 µA; n = 8). The antidromic latency and minimal threshold of the cell, for which an axonal branch was found in the ventral reticular formation, was 12 ms and 16 µA, respectively.

Five of the six cells tested for projections to the dorsomedial medulla were silenced during the carbachol-induced REM sleep-like episodes; the remaining one, a cell for which no axonal projections were found, decreased its firing rate from 3.3 to 0.37 Hz.

We searched for the projections of four A5 cells in the region of the RVLM. Two cells were activated antidromically, and each had two axonal branches (26 ms and 18 µA and 12 ms and 34 µA for one cell; 8 ms and 57 µA and 8.3 ms and 76 µA for the other, respectively) (Fig. 5D). Three of these cells were silenced by carbachol, and the activity of the fourth (which had projections to the RVLM) decreased from 3.6 to 0.3 Hz.

We also found five silent cells in the A5 region that could be activated with fixed latencies and distinct thresholds from the XII nucleus, suggesting that the responses were antidromic. Due to the absence of spontaneous activity, collision tests could not be performed. The presumed antidromic latencies of these cells averaged 2.9 ± 1 ms (range: 1.7-4.0 ms), significantly shorter than the shortest latencies of the spontaneously active A5 cells that fulfilled our criteria for noradrenergic neurons (P < 0.01).

Effects of bilateral inhibition of A5 cell activity by clonidine on XII nerve activity. Our findings indicated that the putative noradrenergic cells of the A5 region are spontaneously active under the control conditions of our experiments and become silent, or nearly silent, during carbachol-induced REM sleep-like episodes. The data also suggested that these cells had few or no axonal arborizations within the XII nucleus. Because the latter finding contrasted with retrograde tracing studies showing that A5 neurons project to the XII nucleus (1, 23), we considered the possibility that our study missed A5 cells with such projections.

To determine the impact of the simultaneous silencing of many A5 cells on XII nerve activity, we microinjected clonidine directly into the A5 region on both sides (Fig. 6). In eight rats, 20-nl injections of 0.75 mM clonidine were made first ipsilateral and then, 7.3 ± 1 min later, contralateral to the recorded XII nerve activity. The amount of clonidine per side was 1:1,000 of the amount sufficient to silence A5 neurons by systemic administration, with the volume selected to occupy a sphere of tissue having a diameter of 0.5 mm before diffusion (33, 38). No change in XII activity or blood pressure occurred within 3-5 min after either uni- or bilateral injections. After both injections, the level of XII nerve activity was not significantly different from the activity before the injections (106 ± 14% of control, P = 0.6; n = 8). The respiratory rate also did not change: it was 46.5 ± 4.4 breaths/min before and 46.1 ± 5.0 breaths/min after the injections (P = 0.6; n = 8). There were no changes in arterial blood pressure (89 ± 17 mmHg before, and 87 ± 14 mmHg after the injections). The effectiveness of clonidine injections into A5 was confirmed in a preliminary study (37) in which successive injections into the A5 and then LC triggered responses whose pattern and duration were similar to the REM sleep-like episodes elicited by carbachol injections into the pontine tegmentum, whereas bilateral clonidine injections into LC alone did not have such an effect.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   Distribution of the sites where clonidine was bilaterally microinjected (0.75 mM, 20 nl each injection) to inhibit the activity of noradrenergic A5 neurons. All sites were localized within the same region where we recorded A5 cells physiologically identified as noradrenergic (compare with Fig. 1B). The sites are superimposed on a standard brain section taken from Paxinos and Watson (41).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We determined that putative noradrenergic cells of the A5 group are silenced during REM sleep-like episodes elicited by small pontine injections of carbachol. This is similar to the behavior of LC and sub-LC cells during both natural REM sleep (6, 43) and the REM sleep-like condition elicited by carbachol (21) and suggests that A5 neurons are also silenced during natural REM sleep. The A5 neurons studied included those with identified axonal projections to the RVLM and NTS. Thus REM sleep is associated with silencing of noradrenergic A5 neurons and, presumably, a reduction of norepinephrine release in the NTS and RVLM (and probably also in the region of spinal preganglionic sympathetic, dorsal horn sensory neurons and other major projection sites of A5 neurons) (8, 9, 26). Because we found no A5 cells with axonal projections to the XII motor nucleus, the contribution of these cells to the noradrenergic excitation of XII motoneurons appears to be negligible.

The focus of this study was on noradrenergic A5 neurons and their behavior during REM sleep. To ensure that we recorded from noradrenergic cells, we used a combination of criteria (see METHODS) previously established for the identification of noradrenergic A5 neurons (26) and the prototypic noradrenergic neurons of the LC (32, 39, 42). We also verified that our recording sites were localized among tyrosine hydroxylase-containing neurons of the A5 region. Thus we maximized the probability that the cells studied were noradrenergic. The A5 cell activity decreased consistently during the time when carbachol elicited REM sleep-like signs, suggesting that the population of cells recorded in this study was homogeneous.

The function(s) of A5 neurons is not well established, but their axonal projections suggest a major involvement of the A5 group in cardiorespiratory regulation and/or nociception (8, 9, 26, 48). Recordings from these cells and local lesions or suppression of neuronal activity in the A5 region with muscimol show that they mediate a part of sympathoexcitatory response to stimulation of arterial chemoreceptors (30, 31; see, however, Ref. 47) and posthypoxic respiratory changes (10). In the neonatal rat brain stem in vitro, local application of clonidine into the A5 region results in slowing of respiratory rate (15, 28). Neuroanatomic studies show that descending projections of A5 cells include the dorsal horn and intermediolateral cell column in the spinal cord, ventrolateral medulla (RVLM as well as the pre-Bötzinger region containing neurons important for respiratory rhythmogenesis), NTS (a viscerosensory nucleus), and brain stem motor nuclei (1, 8, 9, 23, 26, 48). However, similar to our results with clonidine microinjections, acute inhibition of cells within the A5 region with muscimol does not alter the resting arterial blood pressure or sympathetic activity in anesthetized rats (25). Similarly, arterial blood pressure and sympathetic responses to stimulation of the RVLM are not altered in anesthetized rats following noradrenergic cell-specific destruction of A5 neurons (44). On the other hand, microinjections of glutamate, which stimulates noradrenergic and other cells in the A5 region, reduce the respiratory rate and increase arterial blood pressure (13, 26). These effects may be due to activation of silent cells in the A5 region. Indeed, we found silent cells in the A5 region that may project to the dorsomedial medulla but, based on their fast conduction velocities, were not noradrenergic.

Noradrenergic neurons may suppress nociceptive transmission in the spinal cord (9, 48). Because A5 neurons project to the dorsal horn of the spinal cord, their silencing during REM sleep, along with the silencing of other noradrenergic neurons, may explain the increase in the size of the receptive field of dorsal horn neurons during this state (29).

Neuroanatomic data also show that noradrenergic A5 neurons project to cranial motor nuclei. According to Grzanna et al. (23), A5 and A7 neurons are the main source of noradrenergic innervation of the facial and trigeminal motor nuclei, whereas Aldes et al. (1) estimated that A5 cells represent 10-20% of pontine noradrenergic cells with projections to the XII nucleus. Many other studies show that cells in the A5 region project to the XII motor nucleus (e.g., Refs. 14, 17), although without identification of such cells as noradrenergic. Based on the existing data, it is impossible to determine whether our inability to find at least one putative noradrenergic cell projecting to the XII nucleus out of six tested is sufficient to conclude that these cells do not project to the XII nucleus. It is, however, well established that many noradrenergic fibers are present in the XII nucleus (2, 3). Thus, to reconcile these data with our difficulty in finding A5 cells antidromically activated from the XII nucleus, we need to assume that there are relatively few A5 neurons projecting to the nucleus. For such few cells to have significant impact on the activity of XII motoneurons, their axons would have to possess extensive arborizations within the XII nucleus. If so, we apparently missed such cells in our study.

An alternative explanation allowing us to reconcile the apparent lack of A5 cell projections to the XII nucleus with their hypothesized direct involvement in the control of XII motoneurons would be that the noradrenergic A5 cells projecting to the NTS (three out of five cells tested had projections) act on XII motoneuronal dendrites that extend into the NTS and in other directions (4).

To assess the role of A5 cells in the control of XII motoneuron activity, we performed bilateral microinjections of clonidine into the A5 region. Local application of clonidine inhibits noradrenergic A5 neurons (15, 26, 28). Because norepinephrine excites XII motoneurons (5, 22, 40, 49), the silencing of A5 neurons by clonidine should reduce XII nerve activity. However, no changes occurred, suggesting that silencing A5 neurons (and presumed decrement in norepinephrine release in their target areas) has no effect on the activity of XII motoneurons. This contrasts with the profound depression of XII nerve activity during the REM sleep-like episodes produced by pontine carbachol in our experiments, suggesting that the two phenomena are not causally related.

Thus, although a reduced release of norepinephrine was observed in the XII nucleus region during the REM sleep-like suppression of motor activity (35), our results show that few or no A5 cell projections target XII motoneurons and the activity of A5 cells makes little or no contribution to the activity of XII motoneurons under our experimental conditions. By exclusion, norepinephrine cells of the A7 group and/or sub-LC region may play a larger role in withdrawing noradrenergic excitation from XII motoneurons during REM sleep, whereas the silencing of A5 neurons may be important in regulating afferent transmission in the NTS and elsewhere. Our recent preliminary data suggest that the silencing of A5 neurons plays a facilitatory role in the generation of the REM sleep-like signs in anesthetized rats (37). Thus A5 neurons may be an important part of the brain stem network generating REM sleep. This function has not been considered but is consistent with the extensive projections of A5 neurons to brain stem and hypothalamic sites involved in the regulation of sleep (8).


    ACKNOWLEDGEMENTS

The authors thank Dr. Patrice G. Guyenet for critical comments on an earlier version of the manuscript.


    FOOTNOTES

The study was supported by Specialized Center of Research Grant HL-60287. P. Janssen was supported by Training Grant HL-07713.

Address for reprint requests and other correspondence: V. B. Fenik, Dept. of Animal Biology 205E/VET, School of Veterinary Medicine, Univ. of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6046 (E-mail: vfenik{at}vet.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

June 14, 2002;10.1152/japplphysiol.00225.2002

Received 15 March 2002; accepted in final form 5 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aldes, LD, Chapman ME, Chronister RB, and Haycock JW. Sources of noradrenergic afferents to the hypoglossal nucleus in the rat. Brain Res Bull 29: 931-942, 1992.

2.   Aldes, LD, Chronister RB, Shelton CI, Haycock JW, Marco LA, and Wong DL. Catecholamine innervation of the rat hypoglossal nucleus. Brain Res Bull 21: 305-312, 1988.

3.   Aldes, LD, Shaw B, Chronister RB, and Haycock JW. Catecholamine-containing axon terminals in the hypoglossal nucleus of the rat: an immuno-electronmicroscopic study. Exp Brain Res 81: 167-178, 1990.

4.   Altschuler, SM, Bao X, and Miselis RR. Dendritic architecture of hypoglossal motoneurons projecting to extrinsic tongue musculature in the rat. J Comp Neurol 342: 538-550, 1994.

5.   Al-Zubaidy, ZA, Erickson RL, and Greer JJ. Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats. Pflügers Arch 431: 942-949, 1996.

6.   Aston-Jones, G, and Bloom FE. Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. J Neurosci 1: 876-886, 1981.

7.   Baghdoyan, HA. Cholinergic mechanisms regulating REM sleep. In: Integrating Basic Research and Clinical Practice, edited by Schwartz WJ.. Basel: Karger, 1997, p. 88-116.

8.   Byrum, CE, and Guyenet PG. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J Comp Neurol 261: 529-542, 1987.

9.   Clark, FM, and Proudfit HK. The projections of noradrenergic neurons in the A5 catecholamine cell group to the spinal cord in the rat: anatomical evidence that A5 neurons modulate nociception. Brain Res 616: 200-210, 1993.

10.   Coles, SK, and Dick TE. Neurones in the ventrolateral pons are required for post-hypoxic frequency decline in rats. J Physiol 497: 79-94, 1996.

11.   Dampney, RAL Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74: 323-364, 1994.

12.   Davies, RO, and Kubin L. Projection of pulmonary rapidly adapting receptors to the medulla of the cat: an antidromic mapping study. J Physiol 373: 63-86, 1986.

13.   Dawid-Milner, MS, Lara JP, Gonzaléz-Barón S, and Spyer KM. Respiratory effects of stimulation of cell bodies of the A5 region in the anaesthetised rat. Pflügers Arch 441: 434-443, 2001.

14.   Dobbins, EG, and Feldman JL. Differential innervation of protruder and retractor muscles of the tongue in rat. J Comp Neurol 357: 376-394, 1995.

15.   Errchidi, S, Hilaire G, and Monteau R. Permanent release of noradrenaline modulates respiratory frequency in the newborn rat: an in vitro study. J Physiol 429: 497-510, 1990.

16.   Fay, R, and Kubin L. Pontomedullary distribution of 5-HT2A receptor-like protein in the rat. J Comp Neurol 418: 323-345, 2000.

17.   Fay, RA, and Norgren R. Identification of rat brain stem multisynaptic connections to the oral motor nuclei using pseudorabies virus. III. Lingual muscle motor systems. Brain Res Rev 25: 291-311, 1997.

18.   Fenik, V, Fenik P, and Kubin L. A simple cuff electrode for nerve recording and stimulation in acute experiments on small animals. J Neurosci Methods 116: 147-150, 2001.

19.   Fenik, V, Janssen P, Davies RO, and Kubin L. Activity of noradrenergic pontine A5 neurons is suppressed during REM sleep-like episodes produced by carbachol in urethane-anesthetized rats (Abstract). Soc Neurosci Abstr 26: 2026, 2000.

20.   Fenik, V, Marchenko V, Davies RO, and Kubin L. Noradrenergic A5 neurons with axonal projections to the nucleus of the solitary tract (NTS) are silenced during carbachol-induced REM sleep-like episodes in anesthetized rats (Abstract). Soc Neurosci Abstr 27 (836): 18, 2001.

21.   Fenik, V, Ogawa H, Davies RO, and Kubin L. The activity of locus coeruleus neurons is reduced in urethane-anesthetized rats during carbachol-induced episodes of REM sleep-like suppression of upper airway motor tone (Abstract). Soc Neurosci Abstr 25: 2144, 1999.

22.   Funk, GD, Smith JC, and Feldman JL. Development of thyrotropin-releasing hormone and norepinephrine potentiation of inspiratory-related hypoglossal motoneuron discharge in neonatal and juvenile mice in vitro. J Neurophysiol 72: 2538-2541, 1994.

23.   Grzanna, R, Chee WK, and Akeyson EW. Noradrenergic projections to brain stem nuclei: evidence for differential projections from noradrenergic subgroups. J Comp Neurol 263: 76-91, 1987.

24.   Guyenet, PG. Neural structures that mediate sympathoexcitation during hypoxia. Respir Physiol 121: 147-162, 2000.

25.   Guyenet, PG, and Koshiya N. Respiratory-sympathetic integration in the medulla oblongata. In: Central Neural Mechanisms in Cardiovascular Regulation, edited by Kunos G, and Ciriello J.. Boston, MA: Birkhauser, 1991, vol. 2, p. 226-247.

26.   Huangfu, DH, Koshiya N, and Guyenet PG. A5 noradrenergic unit activity and sympathetic nerve discharge in rats. Am J Physiol Regul Integr Comp Physiol 261: R393-R402, 1991.

27.   Jodkowski, JS, Coles SK, and Dick TE. Prolongation in expiration evoked from ventrolateral pons of adult rats. J Appl Physiol 82: 377-381, 1997.

28.   Johnson, SM, Smith JC, and Feldman JL. Modulation of respiratory rhythm in vitro: role of Gi/o protein-mediated mechanisms. J Appl Physiol 80: 2120-2133, 1996.

29.   Kishikawa, K, Uchida H, Yamamori Y, and Collins JG. Low-threshold neuronal activity of spinal dorsal horn neurons increases during REM sleep in cats: comparison with effects of anesthesia. J Neurophysiol 74: 763-769, 1995.

30.   Koshiya, N, and Guyenet PG. Role of the pons in the carotid sympathetic chemoreflex. Am J Physiol Regul Integr Comp Physiol 267: R508-R518, 1994.

31.   Koshiya, N, and Guyenet PG. A5 noradrenergic neurons and the carotid sympathetic chemoreflex. Am J Physiol Regul Integr Comp Physiol 267: R519-R526, 1994.

32.   Koyama, Y, Jodo E, and Kayama Y. Sensory responsiveness of "broad-spike" neurons in the laterodorsal tegmental nucleus, locus coeruleus and dorsal raphe of awake rats: implications for cholinergic and monoaminergic neuron-specific responses. Neuroscience 63: 1021-1031, 1994.

33.   Kubin, L. Carbachol models of REM sleep: recent developments and new directions. Arch Ital Biol 139: 147-168, 2001.

34.   Kubin, L, Davies RO, and Pack AI. Control of upper airway motoneurons during REM sleep. News Physiol Sci 13: 91-97, 1998.

35.   Lai, YY, Kodama T, and Siegel J. Changes in monoamine release in the ventral horn and hypoglossal nucleus linked to pontine inhibition of muscle tone: an in vivo microdialysis study. J Neurosci 21: 7384-7391, 2001.

36.   Levitt, P, and Moore RY. Origin and organization of brain stem catecholamine innervation in the rat. J Comp Neurol 186: 505-528, 1979.

37.   Marchenko, V, Fenik V, Davies RO, and Kubin L. Clonidine microinjections into noradrenergic (NA) A5 and locus coeruleus regions elicit REM sleep-like effects in urethane-anesthetized rats (Abstract). Soc Neurosci Abstr 27 (524): 15, 2001.

38.   Nicholson, C. Diffusion from an injected volume of a substance in the brain tissue with arbitrary volume fraction and tortuosity. Brain Res 333: 325-329, 1985.

39.   Page, ME, and Valentino RJ. Locus coeruleus activation by physiological challenges. Brain Res 35: 557-560, 1994.

40.   Parkis, MA, Bayliss DA, and Berger AJ. Actions of norepinephrine on rat hypoglossal motoneurons. J Neurophysiol 74: 1911-1919, 1995.

41.   Paxinos, G, and Watson C. The Rat Brain in Stereotaxic Coordinates (2nd ed.). San Diego, CA: Academic, 1991.

42.   Reiner, PB. Clonidine inhibits central noradrenergic neurons in unanesthetized cats. Eur J Pharmacol 115: 249-257, 1985.

43.   Reiner, PB. Correlational analysis of central noradrenergic neuronal activity and sympathetic tone in behaving cats. Brain Res 378: 86-96, 1986.

44.   Schreihofer, AM, Stornetta RL, and Guyenet PG. Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat. J Physiol 529: 221-236, 2000.

45.   Spyer, KM. Central nervous mechanisms contributing to cardiovascular control. J Physiol 474: 1-19, 1994.

46.   Sun, MK. Central neural organization and control of sympathetic nervous system in mammals. Prog Neurobiol 47: 157-233, 1995.

47.   Sun, MK, and Reis DJ. Urethane directly inhibits chemoreflex excitation of medullary vasomotor neurons in rats. Eur J Pharmacol 293: 237-243, 1995.

48.   Tavares, I, Lima D, and Coimbra A. The pontine A5 noradrenergic cells which project to the spinal cord dorsal horn are reciprocally connected with the caudal ventrolateral medulla in the rat. Eur J Neurosci 9: 2452-2461, 1997.

49.   White, SR, Fung SJ, Jackson DA, and Imel KM. Serotonin, norepinephrine and associated neuropeptides: effects on somatic motoneuron excitability. Prog Brain Res 107: 183-199, 1996.

50.   Woch, G, Ogawa H, Davies RO, and Kubin L. Behavior of hypoglossal inspiratory premotor neurons during the carbachol-induced, REM sleep-like suppression of upper airway motoneurons. Exp Brain Res 130: 508-520, 2000.

J APPL PHYSIOL 93(4):1448-1456
8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Physiol.Home page
J. W. Lu, V. B. Fenik, J. L. Branconi, G. L. Mann, I. Rukhadze, and L. Kubin
Disinhibition of perifornical hypothalamic neurones activates noradrenergic neurones and blocks pontine carbachol-induced REM sleep-like episodes in rats
J. Physiol., July 15, 2007; 582(2): 553 - 567.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
E. Chan, H. W. Steenland, H. Liu, and R. L. Horner
Endogenous Excitatory Drive Modulating Respiratory Muscle Activity across Sleep-Wake States
Am. J. Respir. Crit. Care Med., December 1, 2006; 174(11): 1264 - 1273.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. Kubin, G. F. Alheid, E. J. Zuperku, and D. R. McCrimmon
Central pathways of pulmonary and lower airway vagal afferents
J Appl Physiol, August 1, 2006; 101(2): 618 - 627.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
V. B. Fenik, R. O. Davies, and L. Kubin
REM Sleep-like Atonia of Hypoglossal (XII) Motoneurons Is Caused by Loss of Noradrenergic and Serotonergic Inputs
Am. J. Respir. Crit. Care Med., November 15, 2005; 172(10): 1322 - 1330.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
V. A Bouryi and D. I Lewis
The modulation by 5-HT of glutamatergic inputs from the raphe pallidus to rat hypoglossal motoneurones, in vitro
J. Physiol., December 15, 2003; 553(3): 1019 - 1031.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
93/4/1448    most recent
00225.2002v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fenik, V.
Right arrow Articles by Kubin, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fenik, V.
Right arrow Articles by Kubin, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online