Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bou-Flores, Céline and
Albert J. Berger.
Gap Junctions and Inhibitory Synapses Modulate Inspiratory
Motoneuron Synchronization.
J. Neurophysiol. 85: 1543-1551, 2001.
Interneuronal electrical coupling via gap
junctions and chemical synaptic inhibitory transmission are known to
have roles in the generation and synchronization of activity in
neuronal networks. Uncertainty exists regarding the roles of these two modes of interneuronal communication in the central respiratory rhythm-generating system. To assess their roles, we performed studies
on both the neonatal mouse medullary slice and en bloc brain
stem-spinal cord preparations where rhythmic inspiratory motor activity
can readily be recorded from both hypoglossal and phrenic nerve roots.
The rhythmic inspiratory activity observed had two temporal
characteristics: the basic respiratory frequency occurring on a long
time scale and the synchronous neuronal discharge within the
inspiratory burst occurring on a short time scale. In both
preparations, we observed that bath application of gap-junction blockers, including 18-glycyrrhetinic acid, 18
-glycyrrhetinic acid, and carbenoxolone, all caused a reduction in respiratory frequency. In contrast, peak integrated phrenic and hypoglossal inspiratory activity was not significantly changed by gap-junction blockade. On a short-time-scale, gap-junction blockade increased the
degree of synchronization within an inspiratory burst observed in both
nerves. In contrast, opposite results were observed with blockade of
GABAA and glycine receptors. We found that
respiratory frequency increased with receptor blockade, and
simultaneous blockade of both receptors consistently resulted in
a reduction in short-time-scale synchronized activity observed in
phrenic and hypoglossal inspiratory bursts. These results support the
concept that the central respiratory system has two components: a
rhythm generator responsible for the production of respiratory cycle
timing and an inspiratory pattern generator that is involved in
short-time-scale synchronization. In the neonatal rodent, properties of
both components can be regulated by interneuronal communication via gap
junctions and inhibitory synaptic transmission.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has been
proposed that rhythmic activity in central neural networks can arise
from both interneuronal gap-junction-mediated electrical communication
and inhibitory synaptic transmission (Tamas et al. 2000;
Zhang et al. 1998
). One of the most fundamental biological rhythms is the respiratory rhythm. In the rodent, the pre-Bötzinger complex (PBC) located in the ventrolateral medulla is now generally accepted as the site where respiratory rhythm is
generated (Ballanyi et al. 1999
; Koshiya and
Smith 1999
; Rekling and Feldman 1998
). This
structure is likely responsible for the long-time-scale features of
respiratory cycle timing, i.e., respiratory frequency. Yet inspiratory
motor activity is also associated with short-time-scale neuronal
synchrony that occurs within an inspiratory burst (Cohen et al.
1987
; Sica et al. 1991
). Little is known about mechanisms that modulate this short-time-scale synchrony and how such
modulation might alter respiratory cycle timing.
There now exist two very powerful in vitro mammalian preparations for
studying the properties of respiratory rhythm and neural activity. One
preparation, originally developed by Suzue (Suzue et al.
1983), uses the brain stem-spinal cord from the neonatal rodent, termed the en bloc preparation. Using the en bloc preparation, rhythmic inspiratory phase activity can be recorded from numerous motor
nerves including the phrenic and hypoglossal nerves. The second
preparation is the medullary slice preparation developed by
Smith and colleagues (1991)
, where rhythmic inspiratory
activity can readily be recorded from the hypoglossal nerve roots. We
used both of these preparations to investigate the role played by gap junctions and inhibitory synaptic transmission in the modulation of
inspiratory phase activity.
Gap-junction communication has been shown to be important in a number
of neuronal systems, including spontaneous bursting in the embryonic
retina (Wong et al. 1998), synchronization within the
neocortex (Galarreta and Hestrin 1999
; Gibson et
al. 1999
), and motor pattern coordination within the neonatal
spinal cord (Tresch and Kiehn 2000
). A preliminary
report (O'Neal et al. 1999
) showed the presence of
gap-junction-related proteins (connexins) in neurons of the PBC, and
thus gap-junction communication could potentially be important for
their function. We thus decided to investigate whether blocking of gap
junctions has effects on respiratory rhythm generation and the
synchronous activity observed within inspiratory motor bursts. In
recent years, a number of relatively specific gap-junction blockers
have been developed. These include glycyrrhetinic acid derivatives and
related compounds including: 18
-glycyrrhetinic acid (18
-GA),
18
-glycyrrhetinic acid (18
-GA), and carbenoxolone (CBX)
(Davidson and Baumgarten 1988
; Davidson et al.
1986
; Goldberg et al. 1996
). In the experiments
described, we tested the effects of these gap-junction blockers on
inspiratory neural activity.
A number of studies have shown that inhibitory interneurons are also
involved in the synchronization of neuronal systems (Cobb et al.
1995; Tamas et al. 2000
; Zhang et al.
1998
). Yet the role of GABAA and glycine
receptor-mediated synaptic transmission in the generation of rhythmic
respiratory activity and synchronization of inspiratory motor activity
is not fully understood (Ballanyi et al. 1999
;
Ramirez and Richter 1996
; Rekling and Feldman
1998
). Depending on the type of preparation and its age,
blockade of GABAergic and glycinergic synaptic transmission can produce
differing effects on respiratory motor outflow (Pierrefiche et
al. 1998
; Ramirez et al. 1996
). Also, in vivo
experiments have shown that intraventricular administration of
bicuculline to block GABAA receptors and
strychnine to block glycine receptors altered the degree of synchrony
in phrenic nerve activity (Schmid and Böhmer 1989
). Thus in this study, we tested the effects of blockade of either or both GABAA receptors and glycine
receptors on respiratory rhythm and synchronous activity within an
inspiratory burst.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparations
In vitro experiments were performed on either rhythmically active brain stem-spinal cord (en bloc) or medullary slice preparations from Swiss Webster mice (P1-5).
The methods used are fully described elsewhere (Bou-Flores et
al. 2000; Gibson and Berger 2000
; Hilaire
et al. 1997a
,b
). In brief, for both preparations, mice were
anesthetized deeply with halothane. For the en bloc preparation, the
medulla and the cervical spinal cord were isolated and removed from the
animal and then superfused at room temperature with an artificial
cerebrospinal fluid (ACSF; see following text for composition). For
slice studies, the medulla was first isolated, and then a transverse
slice (500- to 700-µm thick) was cut at the level of nucleus ambiguus
with a vibratome. This slice, including the most rostral hypoglossal nerve rootlets, was then placed into a recording chamber and superfused for at least 30 min with a high K+ ACSF solution
(see following text for composition) before making recordings.
Recording
For both preparations, the temperature of the recording chamber was maintained between 27 and 28°C. Glass suction electrodes, filled with ACSF, were used to record from the cut ends of the C4 phrenic rootlets (en bloc preparation) and the cut ends of the hypoglossal rootlets (medullary slice and en bloc preparations). Raw nerve signals were amplified using a CyberAmp 320 (Axon Instruments) and band-pass filtered from 10 Hz to 10 kHz. In addition, the filtered signal was rectified and integrated using a custom built "leaky" integrator with a time constant of 50 ms. Both the filtered and integrated signals were digitized (Neuro-Corder, Neurodata Instruments), stored on a computer hard disk for off-line analysis, and displayed on a chart recorder (Gould TA-2000). To make comparisons with the data obtained from the medullary slice and en bloc preparations, the experimental procedures, pharmacological studies, and data-compilation methods used in both were identical.
Solutions and drug application
The normal ACSF contained (in mM):118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, and 30 D-glucose and was gassed with 95%
O2-5% CO2. The high
K+ ACSF contained (in mM) 110 NaCl, 9 KCl, 1.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, and 30 D-glucose and was gassed with 95%
O2-5% CO2. The osmolarity
of the ACSFs was 310 mOsm, and pH was adjusted to 7.4 by NaOH. All
drugs used in this study were diluted from stock solutions in ACSF and
superfused over the preparations at a rate of 3-4 ml/min. In
experiments with both preparations, we added gap-junction blockers to
the superfusate for a 20-min period. The following gap-junction
blockers, with their concentrations indicated, were tested:
carbenoxolone (CBX; Sigma), 100 µM; 18-glycyrrhetinic acid
(18
-GA; Sigma), 50 µM; and 18
-glycyrrhetinic acid (18
-GA; Sigma), 50 µM. In experiments to block GABAergic and glycinergic synaptic transmission, we added to the superfusate bathing the preparations bicuculline methiodide (5 µM, Sigma) and strychnine hydrochloride (1 µM, Sigma), respectively.
Data analysis
In both preparations, the number of integrated bursts of activity was measured every minute during a period of at least 5 min prior to any drug application to estimate the mean control respiratory frequency. Drugs were then applied and the resulting changes were expressed as percentage of the control values.
For analysis of the "leaky" integrated nerve activity, we measured its average peak amplitude and integrated area based on averaging approximately 30 rectified-integrated bursts of activity. During the control period, the data were taken just prior to drug application. During drug application, the data were derived from the period that began after the onset of the effect on respiratory frequency and always ended at the point where drug application stopped. During wash periods, data were taken after 30 min of washing.
To analyze synchronous activity during an inspiratory burst, we computed an average power spectrum (Clampfit Version 8.0, Axon Instruments) that was derived from an analysis of 10 inspiratory bursts. The data during control were derived from the period just prior to drug application. During drug application, the 10 inspiratory bursts were from the period just prior to the point where drug application stopped. This period typically was 18-20 min after the start of drug application. During wash periods, data were taken after 30 min of washing. For the power spectrum analysis, the filtered nerve signal was further band-pass filtered from 1 to 200 Hz, and using a sampling rate of 2,000 Hz, a power spectrum was computed based on 512 data points. The spectral resolution was therefore 3.91 Hz/bin. The sampling period for the power spectral analysis commenced at the start of each inspiratory burst. The average power spectrum was computed in one of two ways. First, and in all cases, we computed an average power spectral density (PSD) based on the absolute power (µV2) of the signal in each frequency bin. Second, when comparing the PSDs between nerves (see Fig. 6 for example), we computed the relative power (absolute power in each bin/total power) where the total power is the absolute power in each bin summed over all frequencies in the power spectrum.
All the results are expressed as percentage of the control values. All values are given as means ± SE. Statistical analysis was performed with a two-tailed Student's t-test, and significance was assumed if P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In both neonatal mouse en bloc and medullary slice preparations,
the central respiratory network continues to produce rhythmic inspiratory bursts as observed in the phrenic (en bloc) and hypoglossal (slice) nerve roots. These exhibit little variability in burst frequency and amplitude. In the en bloc preparations (n = 30), we found the mean control frequency and mean duration of the
inspiratory bursts (TI) to be 5.0 ± 0.5 min1 and 867 ± 44 ms, respectively. In the medullary slice preparations (n = 20), we found the mean control frequency and mean
TI to be 5.4 ± 0.4 min
1 and 858 ± 40 ms, respectively. The mean control frequency and mean TI were not
significantly different between the two preparations. Therefore we
combined the data for both preparations and found the mean control
frequency and mean TI to be 5.2 ± 0.3 min
1 and 862 ± 31 ms, respectively. These values are similar to those published elsewhere
(Hilaire et al. 1997a
,b
; Ramirez et al.
1996
).
Effect of gap-junction blockade on respiratory rhythm
To assess the involvement of gap junctions in modulation of
respiratory rhythm, we added gap-junction blockers to the ACSF superfusing both en bloc and medullary slice preparations. We investigated the effects of various glycyrrhetinic acid compounds that
have been shown to block gap-junction communication by a mechanism that
may involve conformational changes in connexin structure
(Goldberg et al. 1996).
As shown on Fig. 1A, replacing normal ACSF with ACSF containing CBX dramatically decreased respiratory frequency as indicated from recording of phrenic nerve root activity in an en bloc preparation. The effect of CBX was reversible and respiratory frequency recovered within 10-15 min of resuming normal ACSF superfusate. The decrease in respiratory frequency was primarily due to a major increase in expiratory time (TE) rather than a large increase in TI (Fig. 1, B and C).
|
We tested the effects of three different gap-junction blockers on
respiratory frequency in the en bloc preparation. To accomplish this,
each blocker was applied only once to any one preparation, and five
tests were performed with each blocker. We found on average that CBX,
18-GA, and 18
-GA all significantly decreased respiratory frequency by 82 ± 3, 64 ± 8, and 73 ± 8%,
respectively (Fig. 2A). For
all gap-junction blockers, respiratory frequency recovered 10-15 min
after removal of these blockers from the superfusate.
|
We next tested the effect of CBX in the medullary slice preparation since in the en bloc experiments this compound had the greatest effect. In medullary slice preparations CBX (n = 5) also significantly reduced the respiratory frequency by 50 ± 8% (Fig. 3A). Recovery was observed when CBX was removed from the superfusate in a similar time course as that observed with the en bloc preparations. Structural differences between en bloc and medullary slice preparations may be responsible for the quantitative difference in the effect of CBX on respiratory frequency.
|
Effect of gap-junction blockade on inspiratory activity
Next we studied the effects of gap-junction blockers on
inspiratory burst activities in the en bloc preparation by measuring both the area and peak amplitude of the integrated phrenic activity. In
the en bloc preparation, the gap-junction blockers 18-GA and 18
-GA did not significantly affect the area and peak amplitude of
the phrenic burst activity (Fig. 2, B and C). CBX
did not significantly alter the peak amplitude of the integrated
phrenic burst but significantly increased the area (by 150 ± 17%, Fig. 2B). Next we tested the effect of CBX on
hypoglossal activity in the slice. We found that CBX (n = 5) significantly decreased by 36.9 ± 13% the area of integrated hypoglossal bursts (Fig. 3B) but had no
significant effect on the peak amplitude of integrated hypoglossal
inspiratory bursts (Fig. 3C).
We also measured the effect of CBX on TI in both preparations. We found that CBX increased TI of phrenic bursts by 31.6 ± 9% (en bloc) but decreased TI of hypoglossal inspiratory bursts by 20 ± 3% (slice). These contrasting effects of CBX (on TI and area) suggest that structural differences in both preparations may be responsible for these differing effects.
Effect of gap-junction blockade on synchronous activity within an inspiratory burst
We analyzed short-time-scale synchronization of
inspiratory-phase activity in both phrenic and hypoglossal nerves
by performing power spectral analysis of inspiratory activity recorded
in each nerve. In en bloc preparations, application of CBX markedly
enhanced the degree of phrenic nerve synchrony as can be seen in the
raw filtered neurogram shown in Fig.
4A. Power spectral analysis of this activity revealed that in control conditions there was a peak in
the power spectrum between 30 and 40 Hz and that application of CBX
caused a large increase in the amplitude of this spectral peak (Fig.
4B). Removal of CBX from the superfusate resulted in a
partial recovery of the power spectrum (Fig. 4B). We tested the effects of CBX and 18-GA on phrenic nerve synchrony in each of
five en bloc preparations. We found that these gap-junction blockers
caused significant increases (by 112 ± 29 and 110 ± 25%, respectively) in the peak amplitude of the 30- to 40-Hz spectral peak
compared with that observed in control solutions (Fig.
5A).
|
|
In five en bloc preparations, we performed simultaneous recordings from
phrenic and hypoglossal nerve rootlets to determine whether or not
inspiratory-phase synchronization was similar in these two nerve
recordings. While both nerves showed synchronized activity, the
resultant power spectrum for each nerve was different. As shown in Fig.
6A, the peak in the power
spectrum occurred at different frequencies in each nerve. In the en
bloc preparation, we consistently found that the peak of the power
spectrum for the hypoglossal nerve occurred at 10-20 Hz in contrast to
the peak for the phrenic nerve, which occurred at 30-40 Hz. These results suggest that the two different frequencies of synchronization may arise from two separate premotor respiratory central pattern generators that project to the respective motor output systems (Peever and Duffin 2000).
|
In contrast to the preceding results in the en bloc preparation, in the
medullary slice preparation, where only hypoglossal nerve activity is
present, the power spectrum of hypoglossal inspiratory bursts revealed
that two distinct spectral peaks were present, one in the 30- to 40-Hz
range and one in the 10- to 20-Hz range (Fig. 6B). Thus
important differences exist in these two preparations with respect to
synchronized hypoglossal activity that occurs during the inspiratory
burst. Our observation that in the en bloc preparation the hypoglossal
has greater power at lower frequencies compared with the phrenic
spectrum has previously been seen in intact in vivo systems
(Cohen et al. 1987; Sica et al. 1991
).
In five medullary slice preparations, we observed that application of CBX to the superfusion fluid caused the hypoglossal spectral peak at 10-20 Hz to be enhanced by on average 116 ± 18% (Fig. 5B). In contrast, we observed that the spectral peak at 30-40 Hz was not significantly affected by CBX.
Effect of GABAA and glycine receptor antagonists on respiratory rhythm
We next investigated the effect of blocking GABAergic and glycinergic synaptic transmission on respiratory rhythm and on synchronous oscillations of phrenic and hypoglossal nerve activities. The protocol of the experiments performed was similar to that used in the gap-junction blocker experiments describe in the preceding text. In the following experiments, we determined the effects of adding bicuculline (5 µM, 20 min) and strychnine (1 µM, 20 min) to the superfusate bathing en bloc and medullary slice preparations.
In the en bloc preparation, application of bicuculline
(n = 5), strychnine (n = 5), or both
(n = 5) caused a significant enhancement by 78 ± 10, 60 ± 6, and 45 ± 3% of respiratory frequency,
respectively (Fig. 7A). In the
medullary slice preparation (Fig. 7A), similar effects were
observed; respiratory frequency rose significantly by 79 ± 14, 34 ± 7 and 69 ± 15% when the superfusate contained bicuculline (n = 5), strychnine (n = 5), or both (n = 5), respectively. These results in the
mouse on the effect of bicuculline and strychnine on respiratory
frequency are in general agreement with a previous study that used the
rat medullary slice preparation (Shao and Feldman 1997).
|
Effect of GABAA and glycine receptor antagonists on inspiratory activity
In the en bloc preparation, the integrated area of the phrenic
burst (Fig. 7B) was enhanced significantly by 30 ± 5, 31 ± 14, and 25 ± 4% if bicuculline (n = 5), strychnine (n = 5), or both (n = 5)
was added to the superfusate, respectively. In contrast, in the
medullary slice preparation, application of strychnine (n = 5) or bicuculline (n = 5) alone
did not significantly change the integrated hypoglossal area, but
application of both antagonists together significantly increased the
area by 101 ± 11% (n = 5, Fig. 7B).
The latter results are consistent with a previous study in the rat from
our laboratory (Gibson and Berger 2000). We observed that application of both antagonists together significantly decreased by 20 ± 5% the peak integrated of phrenic and hypoglossal
activities (Fig. 7C). In contrast, application of strychnine
alone significantly increased by 27 ± 5% peak integrated phrenic
activity (Fig. 7C). Peak integrated hypoglossal nerve
activity was not altered by application of either antagonist alone
(Fig. 7C).
Since application of bicuculline (n = 5) did not cause any variation in peak phrenic activity (Fig. 7C) but increased the integrated area of the phrenic nerve burst (Fig. 7B), we determined the effect of bicuculline on the TI of the phrenic burst and found an enhancement of 44 ± 9% (data not shown). As expected, since co-application of both antagonists caused an enhancement of the integrated area of activity for both nerves with a reduction in peak activity, we observed that bicuculline enhanced TI of phrenic and hypoglossal activity by 44 ± 9 and 46 ± 8%, respectively (data not shown).
Effect of GABAA and glycine receptor antagonists on synchronous activity within an inspiratory burst
In en bloc preparations, application of bicuculline markedly reduced the degree of phrenic nerve synchrony. Power spectral analysis of nerve activity showed that the 30- to 40-Hz peak in the power spectrum was reduced in amplitude on average by 87 ± 7% (n = 5, Fig. 8). Bicuculline had similar effects on the power spectrum of hypoglossal nerve activity in the medullary slice preparation (Fig. 9). Bicuculline reduced in amplitude both the 10- to 20-Hz and the 30- to 40-Hz peaks by 60 ± 6 and 60 ± 8%, respectively.
|
|
Application of strychnine produced variable effects on the power spectra. The variability was reduced if the population of mice studied was separated into two age groups, P0-1 and P2-4. In the youngest age group, we did not observe a significant effect of strychnine on the power spectrum of the phrenic nerve activity (Fig. 8). In contrast, for the older age group, we observed an 88 ± 3% reduction in the amplitude of the 30- to 40-Hz peak in the phrenic power spectrum (Fig. 8). In contrast to the effects of strychnine on synchronization of phrenic activity in the en bloc preparation, we observed in the medullary slice preparation that in both age groups strychnine application to the superfusate caused an enhancement of the 10- to 20-Hz peak in the power spectrum of hypoglossal nerve activity (Fig. 9). Specifically, strychnine enhanced the amplitude of this peak by 158 ± 64 and 123 ± 23%. Only in the older preparation (P2-4) did we observe that strychnine reduced by 60 ± 5% the 30- to 40-Hz peak in the hypoglossal power spectrum (Fig. 9).
Co-application of both antagonists gave more consistent results. We observed that this caused a decrease by 80 ± 4% in the 30- to 40-Hz spectral peak of phrenic activity (Fig. 8) and by 90 ± 4 and 85 ± 0.5% in the 10- to 20-Hz and 30- to 40-Hz spectral peaks of hypoglossal activity, respectively (Fig. 9).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These experiments have focused on modulation of two different timing characteristics of respiratory motor outflow. One, on a longer time scale associated with respiratory frequency, and the other, on a shorter time scale associated with synchronization of activity within the inspiratory motor burst. The result that gap-junction blockade and inhibitory synaptic transmission antagonism have opposite effects on each of these timing signals provides new information on mechanisms by which these signals can be modulated.
Our primary observations include the findings that gap-junction blockade consistently resulted in a reduction in respiratory frequency, and this occurred in both en bloc and medullary slice preparations. These results are consistent with a role of gap junctions in the generation of respiratory cycle timing. Further, in most cases, gap-junction blockade also caused a marked increase in short-time-scale synchronized activity in both phrenic and hypoglossal inspiratory bursts. This occurred in the absence of a shift in the predominant frequencies in the power spectra of this synchronized activity. In addition, we observed that gap-junction blockade caused minimal or mixed effects on the two measures we used to quantitate amplitude of inspiratory phase motoneuron activity. In contrast to the results with the gap-junction blockers, blockade of GABAA and glycine receptors caused an increase in respiratory frequency. We also found that simultaneous blockade of both of these receptors consistently resulted in a reduction in short-time-scale synchronized activity in both phrenic and hypoglossal inspiratory bursts.
The timing of respiratory activity is thought to arise from the most
upstream element in the respiratory rhythm-generating mechanism,
perhaps involving a network of coupled pacemakers (Ballanyi et
al. 1999; Koshiya and Smith 1999
; Rekling
and Feldman 1998
). The location of this rhythm-generating
structure in the rodent brain stem is in the ventrolateral medulla and
within the PBC. Conceptually it is thought that the respiratory
rhythm-generating system is responsible for the generation of
respiratory frequency. An inspiratory pattern-generating system
responsible for shaping the temporal form of the inspiratory burst is
thought to be downstream of the neural elements responsible for
respiratory rhythm generation (Ballanyi et al. 1999
;
Feldman et al. 1990
). How a unique temporal pattern of
inspiratory drive is directed to each of the various classes of
inspiratory motoneurons (e.g., phrenic, inspiratory hypoglossal and
external intercostal motoneurons) is not known, but may involve a
combination of unique neural elements, circuit connections, and
neuronal properties. Further processing of inspiratory drive may occur
among a set of premotor neurons that then project directly to
motoneurons. Thus electrical and inhibitory chemical synaptic
connections may occur at a number of points in the circuit from the
respiratory rhythm-generating mechanism in the PBC to the inspiratory
motoneurons themselves.
It is currently unknown which structures in the brain stem are
responsible for the generation of inspiratory-phase short time scale
synchronization. Since short-time-scale synchronization has been shown
to be present upstream of respiratory motoneurons (Mitchell and
Herbert 1974; O'Neal et al. 1999
), it is
reasonable to conclude that the respiratory motoneurons themselves are
not the sole source of such synchronization.
Role of gap junctions in inspiratory neural activity
Several studies in neonatal rat and mouse have reported the
presence of electrical coupling in postnatal hypoglossal and other inspiratory brain stem and phrenic motoneurons (Martin-Caraballo and Greer 1999; Mazza et al. 1992
;
Rekling and Feldman 1997
). Electrical coupling is lost
in older motoneurons (Chang et al. 1999
; Mazza et
al. 1992
). It has been suggested that such transient gap-junction communication between motoneurons enhances motoneuron synchronous activity and may function to preserve multiple innervation of single muscle fibers (Balice-Gordon and Lichtman
1994
). We believe it unlikely that our observations of the
effect of gap-junction blockade on respiratory frequency and short time
scale synchronization involve these inter-motoneuronal electrical
communications. Preliminary anatomical data has indicated that
connexins are present in the neonatal and adult rodent PBC
(O'Neal et al. 1999
). Thus the anatomical substrate for
our observed effects of blocking gap junctions, leading to a marked
reduction in respiratory frequency, is likely within the PBC. In light
of these results, it is interesting that models of electrically coupled
oscillatory neurons have shown that a reduction in electrical coupling
can lead to either a decrease or increase in the frequency of slow
oscillations observed in coupled neuronal networks depending on the set
of underlying neuronal voltage- and time-dependent conductances
(Kepler et al. 1990
).
It is our view that short-time-scale synchrony arises from a mechanism
that is different from the one that generates the basic respiratory
cycle frequency, perhaps involving the inspiratory pattern-generator
circuit, premotor neurons, or even electrically coupled neonatal
respiratory motoneurons. Previous studies in the en bloc preparation
using power spectral analysis of both phrenic and cranial nerve
inspiratory phase discharge, as well as inspiratory phase synaptic
current recorded in single phrenic motoneurons, revealed a dominant
spectral peak in a frequency range close to those observed in the
present experiments (Liu et al. 1990; Smith et
al. 1990
). Our results showed that blockade of gap junctions
resulted in an increase in the short-time-scale synchrony observed
during phrenic and hypoglossal motoneuron inspiratory bursts. This
observation may seem counterintuitive because electrical coupling
between neurons is thought to promote not reduce neuronal synchrony.
Yet studies have shown that synchrony in neuronal networks can be
either increased or decreased by electrical coupling
(Marder 1998
). For example, Traub and Wong
(1983)
found that in hippocampal network simulations electrical
coupling between neurons can either increase or decrease
neuronal synchronization. Thus if the electrically coupled neurons act
as an electrical load on a network that is producing synchronized
activity, then the magnitude of synchronization can be reduced. This
idea is supported by our observations that application of gap-junction
blockers to block electrical coupling did not shift the frequency of
the peak in the power spectrum, but increased its amplitude.
Role of GABAA and glycine receptor-mediated synaptic transmission in inspiratory neural activity
There remains considerable controversy regarding the role of
GABAA and glycine receptor-mediated synaptic
transmission in respiratory rhythm generation (Ballanyi et al.
1999; Ramirez and Richter 1996
; Rekling
and Feldman 1998
). The presence of a GABAergic hindbrain
rhythm-generating mechanism during vertebrate embryonic development has
recently been shown (Fortin et al. 1999
). Blockade of
GABAA and glycine receptor-mediated synaptic
transmission results in inconsistent alteration in respiratory motor
outflow that appears to be dependent on the preparation, including the
age of the experimental animal (Pierrefiche et al. 1998
;
Ramirez et al. 1996
). This inconsistency was clearly
seen in our results, as the effects of glycine receptor blockade on the
degree of synchrony were dependent on the postnatal age of the
preparation. Different effects on synchrony also depended on whether
this was studied in the en bloc or medullary slice preparations. In
contrast, we observed consistent results with GABAA receptor blockade or simultaneous blockade
of GABAA and glycine receptor-mediated synaptic
transmission, causing increased respiratory frequency and decreased
synchrony in motor outflow. We observed that
GABAA receptor blockade with bicuculline
consistently reduced the degree of phrenic nerve synchrony recorded in
the en bloc preparation and also both the 10- to 20-Hz and the 30- to
40-Hz peaks observed in hypoglossal activity in the medullary slice
preparation. These results are consistent with computer simulations of
neocortical and hippocampal gamma oscillations (20-80 Hz) that
demonstrated that GABAA-mediated synaptic
transmission was critical to network synchronization (Bush and
Sejnowski 1996
; Wang and Buzsaki 1996
).
It is interesting that in vertebrates during the embryonic period, the
hindbrain shows the development of two types of rhythmic activity that
can be observed in medullary cranial motoneurons. These two rhythmic
activities are characterized by a low and a high frequency,
respectively. It is thought that such primordial rhythm-generating
circuits may evolve into the mature respiratory rhythm-generating
mechanism (Fortin et al. 1999).
GABAA receptor-mediated events have a role in the
high-frequency embryonic rhythms. Blockade of
GABAA receptors with bicuculline abolished the
high-frequency events but did not affect the low-frequency rhythmic
activity (Fortin et al. 1999
). Our results showing that
blockade of GABAA receptor-mediated events with
bicuculline dramatically reduced the power of the synchronous activity
observed during rhythmic inspiratory bursts of both hypoglossal and
phrenic nerve activities appear to extend these previous results from
the embryological period into the postnatal period. Thus the mechanism
responsible for synchronous activity has a common GABAergic component
at all stages of development.
In some neural systems during the postnatal period, activation of
synaptic GABAA and glycine receptors results in
neuronal depolarization. In hypoglossal motoneurons, glycinergic
depolarization is converted to glycinergic hyperpolarization over the
first two weeks of postnatal life (Singer et al. 1998).
This appears to be due to a reduction in the intracellular
Cl
concentration with postnatal development. In
contrast, some neuronal inhibitory systems are mature at birth. For
example, recordings from ventral respiratory group neurons in the en
bloc preparation revealed that GABAA and glycine
receptor mediated inhibitory postsynaptic potentials are
hyperpolarizing in the neonatal rat (Brockhaus and Ballanyi
1998
).
Function of short-time-scale synchronization in inspiratory neural activity
An important issue is what might be the functional role of
short-time-scale synchronization of motoneuronal activity during the
inspiratory burst. One possibility is that the firing rate of
motoneurons may be increased for the same average excitatory presynaptic activity if this presynaptic activity occurs synchronously rather than asynchronously (Murthy and Fetz 1994). A
recent computer modeling study simulating a motor unit pool was used to
compute the muscle force output as a function of mean motoneuron input firing rate under conditions of differing degrees of input synchrony. It was found that for the same mean input firing rate that muscle force
output rose with increased input synchrony (Baker et al. 1999
). Thus short-time-scale synchronization during the
inspiratory motor burst could function to facilitate premotor and
motoneuron activity as well as increase inspiratory muscle force
output. The ability to modulate the degree of synchronization through modulation of neuronal electrical coupling and inhibitory synaptic communication could enhance or reduce motoneuronal activity and thereby
affect inspiratory muscle force.
![]() |
ACKNOWLEDGMENTS |
---|
We thank Dr. J. A. O'Brien and E. E. Eggers for helpful comments on the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-14857 to A. J. Berger.
![]() |
FOOTNOTES |
---|
Address for reprint requests: A. J. Berger, Dept. of Physiology and Biophysics, School of Medicine, University of Washington, Box 357290, Seattle, WA 98195-7290 (E-mail: Berger{at}u.washington.edu).
Received 11 August 2000; accepted in final form 18 December 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|