Vol. 94, Issue 3, 913-922, March 2003
Respiratory response to activation or disinhibition of the
dorsal periaqueductal gray in rats
Linda F.
Hayward,
Camille L.
Swartz, and
Paul W.
Davenport
Department of Physiological Sciences, University of Florida
College of Veterinary Medicine, Gainesville, Florida 32601
 |
ABSTRACT |
The neural substrates mediating
autonomic components of the behavioral defense response have been shown
to reside in the periaqueductal gray (PAG). The cardiovascular
components of the behavioral defense response have been well described
and are tonically suppressed by GABAergic input. The ventilatory
response associated with disinhibition of the dorsal PAG (dPAG) neurons
is unknown. In urethane-anesthetized, spontaneously breathing rats,
electrical stimulation of the dPAG was shown to decrease the expiration
time and increase respiratory frequency, with no change in time of
inspiration. Baseline and the change in diaphragm electromyograph also
increased, resulting in an increase in neural minute activity.
Microinjection of bicuculline methobromide, a
GABAA-receptor antagonist, into the dPAG produced a similar
response, which was dose dependent. Disinhibition of the dPAG also
produced a decrease in inspiration time. These results suggest that
GABAA-mediated suppression of dPAG neurons plays a role in
the respiratory component of behavioral defense responses. The
respiratory change is due in part to a change in brain stem respiratory
timing and phasic inspiratory output. In addition, there is an increase
in tonic diaphragm activity.
control of breathing; hyperventilation; hypertension
| |
INTRODUCTION |
THE DORSAL PERIAQUEDUCTAL
GRAY (PAG; dPAG) nucleus is a relay from various central neural
pathways to the limbic cortex. Stimulation of the dPAG is known to
induce autonomic reflexes that have a strong sympathetic component. The
behavioral response to dPAG stimulation is defensive behavior,
typically characterized by an increase in aggressive or escape actions
of the animal (1-3, 8). Stimulation of this
region of the PAG also elicits fear (15), anxiety
(5), and vocalization (23, 35). Part of the
autonomic response for these behaviors is an increase in muscular, cardiovascular, and ventilatory activity to prepare the animal for
high-intensity exercise. This defensive control system is inhibited by
GABAergic neurons. Injection of GABAA agonists increases the threshold for eliciting defensive behavior, whereas
GABAA antagonist injection into the dPAG elicits
defensive-like behavior (6, 19, 31, 32). Obstruction of
breathing, hypercapnia, bronchoconstriction, and hypoxia elicit
well-characterized respiratory reflexes, and they also elicit defensive
affective responses. The affective components of respiratory behavioral
responses to threatening ventilatory conditions are unknown. It has
been hypothesized that the dPAG is one component of the defensive
behavioral response to ventilatory challenges (9).
The cardiovascular response to dPAG activation has been well
characterized. Chemical activation of the dPAG produces marked increases in arterial pressure and heart rate (HR). Associated with the
hypertension is a general stereotypic pattern of increased blood flow
to skeletal muscle and decreased flow to visceral regions (1, 3,
5). This redistribution of blood flow is also observed when the
animal is paralyzed, indicating that these vasomotor changes are not
secondary to other ventilatory or somatomotor changes (11, 12,
25). Parallel to the increase in blood pressure, an attenuation
of baroreflex function has also been documented during dPAG stimulation
(27, 33). Descending pathways involved in mediating
cardiovascular changes associated with dPAG stimulation include the
ventrolateral PAG (21), the parabrachial nucleus
(27), and sympathoexcitatory neurons in the rostral ventrolateral medulla (33).
The ventilatory response to dPAG stimulation has received very little
investigation. Huang et al. (18) used an excitatory amino
acid receptor agonist, DL-homocysteic acid (DLH), to
stimulate the dPAG while recording diaphragm electromyographic (dEMG)
activity. They reported an increase in respiratory frequency associated with a decrease in both time of inspiration (TI) and time
of expiration (TE) averaged over 10 breaths. There was also
an apparent increase in baseline dEMG activity, but this was not
analyzed. They also reported an increase in nucleus tractus solitarius
inspiratory neuron activity. Although their results provide a
foundation for the study of the respiratory behavior mediated by the
dPAG, the ventilatory pattern response to dPAG stimulation remains
unknown. In addition, it is unknown if the dPAG respiratory behavior is normally inhibited by GABAergic mechanisms.
In the present study, it was hypothesized that stimulation of the dPAG
would increase diaphragm activity, increase respiratory frequency, and
increase neural minute activity. It was further hypothesized that this
respiratory response to dPAG stimulation is normally inhibited by
GABAA. These hypotheses were tested in anesthetized rats.
The respiratory activity was measured from dEMG activity. The
respiratory response to dPAG neural excitation was studied by
electrical stimulation of the nucleus, and dPAG inhibition was studied
by injection of bicuculline methobromide (BicM), a
GABAA-receptor antagonist.
| |
METHODS |
All experiments were performed on adult male Sprague-Dawley rats
(320-420 g) housed in the University of Florida animal care facility. The rats were exposed to a normal 12:12-h light (6 AM to 6 PM)-dark cycle (6 PM to 6 AM). All experimental procedures were
preapproved by the University of Florida Institutional Animal Care and
Use Committee.
General preparation.
Animals were anesthetized with an intraperitoneal injection of urethane
(1.2-1.4 g/kg) and instrumented with femoral arterial and venous
catheters for recording of arterial pressure and administration of
intravenous fluids, respectively. While the animal was in the supine
position, a midline incision was made on the ventral surface of the
neck. A tracheotomy was performed, and the animal was intubated. All
animals were spontaneously breathing a mixture of room air and 100%
oxygen. Two small (0.003-mm-diameter) Teflon-coated, stainless steel
wires with bared tips were inserted on the right side into the costal
region of the diaphragm through the abdominal musculature for
measurement of dEMG activity. The animal was then placed in the
prone position in a stereotaxic head holder (Kopf Instruments, Tugunga,
CA), and the brain region overlying the PAG was exposed by a
limited craniotomy and removal of the dura.
The arterial catheter was attached to a calibrated pressure transducer
connected to an amplifier (Stoelting, Wood Dale, IL). The analog output
from the blood pressure amplifier was connected to a computer
data-sampling system [Cambridge Electronics Design (CED) 1401 computer
interface, Cambridge, UK]. The dEMG electrode wires were connected to
a Grass preamplifier probe (H1P5, Grass Instruments, West Warwick, RI)
in series with a signal amplifier (P511). The dEMG signal was amplified
(5-50 K), bandpass filtered (0.3-3.0 kHz), rectified, and
integrated (Paynter Filter, 50-ms time constant; BAK Electronics,
Rockville, MD). The rectified and integrated dEMG signal was sent to
the CED data-sampling system (Spike2, CED). Resting rectified and
integrated dEMG burst amplitude was arbitrarily adjusted to a value of
1.0-2.0 (arbitrary units) at the beginning of the experiment.
Rectified and integrated dEMG activity and arterial pressure were
recorded simultaneously. Body temperature was monitored continuously
with a rectal temperature probe and maintained within 38 ± 1°C
with a heating blanket (Harvard Bioscience, Holliston, MA).
Supplemental anesthesia was given as necessary throughout the
experiment (0.1 g/kg iv), as evidenced by fluctuations in blood
pressure, HR, or respiration during surgery or in response to a pinch
of the hind paw.
Protocol.
Each rat underwent only one type of PAG stimulation, either electrical
or chemical, following stabilization of resting blood pressure and
respiratory pattern. For electrical stimulation, an insulated,
stainless steel wire monopolar electrode (1 M
) was connected to an
isolated voltage stimulator (DS2A, Digitimer, Hertfordshire, UK), in
series with a programmable stimulator (Master8, AMPI, Jerusalem,
Israel). The monopolar electrode was then secured to a micropositioner
(MP-660, Kopf Instruments, Tujunga, CA) for stereotaxic placement into
the brain. Alternatively, for chemical stimulation, a single-barrel
microinjection pipette, attached to a pressure injection system (PPS-2,
Medical Systems, Greenvale, NY), was secured to the micropositioner.
The pipette or stimulating electrode was positioned into the region of
the dPAG, according to stereotaxic coordinates described by Paxinos and
Watson's The Rat Brain in Stereotaxic Coordinates
(28), between 7.7 and 8.0 mm caudal from bregma,
0.2-0.3 mm lateral from midline, and 3.8 and 4.1 mm ventral to the
surface of the brain.
Electrical stimulation parameters of the dPAG were set between 7 and 10 V, 0.2-ms pulse duration, and 25- to 40-Hz stimulation frequencies.
Continuous high-frequency stimulation was applied for 10-15 s
followed by periods of 30- to 90-s rest. Chemical stimulation of the
dPAG was induced by local microinjection of the
GABAA-receptor antagonist BicM (0.2-1.0 mM; Sigma
Chemical, St. Louis, MO). BicM mediates disinhibition of the dPAG,
uncovering endogenous excitatory inputs (6, 31). BicM was
diluted in artificial cerebrospinal fluid (aCSF) containing 122 mM
NaCl, 3 mM KCl, 25.7 mM NaHCO
, and 1 mM CaCl2, with pH adjusted to 7.4. Small amounts of
fluorescent latex microspheres (Lumafluor, Naples, FL) were mixed into
the aCSF to facilitate later identification of the microinjection
sites. After stereotaxic placement of the microinjection pipette into the left side of the dPAG, 20-65 nl of BicM were microinjected into the brain over 30 s. The ventilatory and cardiovascular
responses were recorded. The volume of microinjectate was determined by carefully monitoring the movement of the meniscus in the microinjection pipette with a monocular microscope equipped with a calibrated eyepiece
(Titan Tools, Buffalo, NY). To control for the effects of
microinjection alone, some animals also received unilateral microinjections of 20-65 nl of aCSF. One minute after completion of a central injection, the pipette was retracted from the brain. Each
animal received only two doses of BicM in a randomized order. A
separate group of control animals received central injections of aCSF.
All microinjections were made at intertrial intervals no less than 30 min.
At the end of the experiment, the animal was euthanized. For those
animals that underwent electrical stimulation, a electrolytic lesion
was made in the stimulation site (10 mA, 10-s duration). For all
animals, the brain was removed and placed in 4% paraformaldehyde solution for 24-72 h. The brains were then frozen to 
16°C, and the midbrain was sliced into 40-µm transverse sections with a cryostat (HM101, Carl Zeiss, Thornwood, NY). The tissue was mounted on
slides and sealed with a coverslip (Antifade, Molecular Probes, Eugene,
OR). Injections and stimulation sites were imaged with a microscope
equipped with both bright field and epifluorescence.
Data analysis.
All data were analyzed off-line by using Spike2 software (CED). Mean
arterial pressure (MAP) was calculated from the difference between the
systolic and diastolic pressures, divided by 3, plus the diastolic
pressure. HR was calculated from the interval between systolic pressure
peaks. The HR and MAP were measured at the end of inspiration.
Respiratory parameters were calculated from individual bursts in the
integrated dEMG signal and then averaged. TI was measured
from the onset of dEMG burst activity to the point at which the peak
dEMG activity began to decline. TE was measured from the
offset of TI to the onset of the following inspiration. Time of the respiratory cycle (Ttot) was determined by calculating the
sum of TI and TE. Baseline dEMG was defined as
the minimum dEMG value measured between bursts. dEMG burst amplitude
was measured as the peak amplitude during TI. The change in
integrated dEMG during inspiration (delta dEMG) was calculated as the
difference between baseline and dEMG peak burst amplitude. Neural
minute activity was calculated by multiplying the delta dEMG amplitude by the instantaneous frequency (14).
For both electrical and chemical stimulation trials, baseline
respiratory and cardiovascular values were averaged over the five-breath period collected just before the onset of stimulation. After the onset of stimulation, cardiorespiratory parameters were then
averaged over successive 10-breath periods. One 10-breath average was
taken during stimulation, referred to as the "stimulation-on average." Immediately after the offset of stimulation, three
10-breath averages were taken (referred to as stimulation-off
averages 1, 2, and 3). During chemical
stimulation, successive 10-breath averages were calculated every
30 s after completion of the microinjection period. A maximum of
eighteen 10-breath averages over ~520-s post-microinjection were measured.
Absolute values for MAP, HR, TI, TE, Ttot, and
respiratory frequency were compared before and after dPAG stimulation.
Peak changes during dPAG stimulation were calculated as the difference between prestimulus vs. poststimulus values. Baseline dEMG, delta dEMG
amplitude, and neural minute ventilation were expressed as a percentage
of control. A one-way ANOVA with repeated measures was used to identify
significant changes in cardiorespiratory parameters after electrical
stimulation of the dPAG. A two-way ANOVA (factors: time and dose) was
used to test for significant dose effects of BicM. A one-way ANOVA was
used to compare the effect of dose on changes in cardiorespiratory
values. When differences were indicated, a Bonferroni/Dunn or Tukey
post hoc comparison was used to identify significant effects.
P < 0.05 was accepted as significant for all tests
used. All data are reported as means ± SE.
| |
RESULTS |
In all animals, the position of the electrical stimulation
electrode was in the dPAG (Fig. 1). The
microinjection site for all animals exposed to BicM or aCSF injection
was also in the dPAG (Fig. 2). The mean
resting MAP, HR, and respiratory frequency of all animals that
underwent electrical stimulation vs. chemical stimulation are shown in
Table 1.
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Composite of electrical stimulation sites (solid ovals) and an
original recording of arterial pressure (AP), heart rate (HR), and
diaphragm electromyographic activity (dEMG) from 1 animal before,
during, and after electrical stimulation of the dorsal periaqueductal
gray (dPAG; right). Left: stimulation sites
(n = 5) were reconstructed from electrolytic lesion
locations. Schematics of midbrain PAG were adapted from Paxinos and
Watson's The Rat Brain in Stereotaxic Coordinates
(28). Nos. to the left indicate approximate
location of brain site relative to bregma in millimeters. dm,
Dorsomedial column of PAG; dl, dorsolateral; lat, lateral; vlat,
ventrolateral; Dr; dorsal raphe; 3mn, third motor nucleus; Su3,
supraoculomotor region. Star indicates location of central aqueduct.
Right: time of electrical stimulation of the dPAG (25 Hz, 8 V, 0.2 ms) is indicated by solid horizontal bar. Arrow from
right panel indicates location of reconstructed stimulation
site. dEMG is represented in arbitrary units (au). bpm,
Beats/min.
|
|
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Composite of chemical (shaded ovals) and artificial cerebrospinal
fluid (asterisks) microinjection sites and an original recording of AP,
HR, and dEMG from 1 animal before, during, and after bicuculline
methobromide (BicM) microinjection into the dPAG (right).
Left: reconstructed microinjection sites (n = 9) are based on the locations of fluorescent microsphere deposits.
Schematics of midbrain PAG were adapted from Paxinos and Watson's
The Rat Brain in Stereotaxic Coordinates (28).
Right: completion of the microinjection of BicM (0.3 mM, 45 nl) into the dPAG (25 Hz, 8 V, 0.2 ms) is indicated by "v." Arrow
from left panel indicates location of reconstructed
microinjection site.
|
|
Electrical stimulation.
Electrical stimulation of the dPAG elicited an immediate increase in
respiratory activity in all animals (Fig. 1). The average change in
respiratory parameters is shown in Figs.
3 and 4.
During electrical stimulation of the dPAG, there was a significant
reduction in both stimulus-on TE and Ttot relative to
control (Fig. 3A). TI did not change
significantly from control during stimulus-on conditions. Associated
with the decrease in TE and Ttot, there was a significant
increase in respiratory frequency (Fig. 3B). At the offset
of stimulation (stimulus-off 1), TE, Ttot, and respiratory frequency were not significantly different from control. Within 30 breaths (stimulus-off 3), TE, Tot, and respiratory
frequency had all returned to control and were significantly different
from the stimulus-on condition. After the offset of stimulation, there was a small reduction in TI, but this change was not
significantly different from control or the stimulus-on condition.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of electrical stimulation on ventilation timing.
A: mean duration of time between inspiratory bursts (Ttot;
 ), time of expiration (TE;
 ), and time of inspiration (TI;
 ) averaged from 5 breaths before (control), 10 breaths
during (Stim on), and 3 successive groups of 10 breath averages each
(Off-1, Off-2, Off-3) after the offset of electrical stimulation of the
dPAG. B: mean respiratory frequency (Resp. Freq.) measured
at control, Stim on, and after electrical stimulation of the dPAG. All
data were averaged from 5 animals, and values are means ± SE.
* Significant difference from control, P < 0.05. # Significant difference between stimulus-off average
and stimulus-on average, P < 0.05.
|
|
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of electrical stimulation on baseline and
inspiratory dEMG and neural minute activity. A: mean
amplitude of baseline dEMG measured as the minimum amplitude between
control, Stim on, and Off-1, Off-2, and Off-3 after the offset of
electrical stimulation of the dPAG. Averages are represented as
percentage of control amplitude within animals (control = 100%).
B: mean change in dEMG inspiratory burst amplitude from
baseline ( dEMG; ) and neural minute activity
() measured at control, Stim on, and after electrical
stimulation of the dPAG. Averages are represented as percentage of
control within animals (control = 100%). All data represent
averages from 5 animals. Values are means ± SE. * Significant
difference from control, P < 0.05. # Significant difference between stimulus-off averages
and stimulus-on average, P < 0.05. Statistical
indicators below symbols correspond to analysis associated with
dEMG.
|
|
Electrical stimulation also elicited an immediate increase in baseline
dEMG activity and peak burst amplitude (Fig. 1). The average increase
in baseline activity during electrical stimulation was significantly
different from control in all animals (Fig. 4A). Immediately
after the offset of stimulation, baseline activity remained high, but
this increase was not significantly different from control. Peak dEMG
burst amplitude increased in the first breath and was sustained
throughout stimulation. The average stimulus-on peak activity was
170 ± 10% above control (P < 0.001). The peak dEMG burst activity was the combination of baseline activity and the
change in dEMG activity with each breath. After the subtraction of
baseline dEMG levels, the average change in dEMG amplitude or delta
dEMG during inspiration showed a significant increase during electrical
stimulation relative to control (Fig. 4B). Immediately after
the offset of stimulation, delta dEMG remained elevated and was not
significantly different from either control or stimulation values.
Delta dEMG returned to control levels and was significantly different
from stimulus-on levels by stimulus-off 2. Associated with the
significant increase in both delta dEMG and respiratory frequency,
there was a significant increase in neural minute activity during the
stimulus-on condition compared with control (Fig. 4B). Immediately after the offset of stimulation, neural minute activity was
not significantly different from the stimulus-on condition but by
stimulus-off 3 had returned to control and was significantly different
from stimulus-on.
Associated with the increase in respiration during electrical
stimulation of the dPAG, there was a significant increase in both MAP
and HR (Table 1). Immediately after the offset of stimulation, MAP and
HR returned to control levels and were not significantly different from control. MAP remained similar to control during stimulus-off 2 and 3. In contrast, during the same time period, HR
dropped significantly below control.
Bicuculline disinhibition.
BicM induced an increase in respiratory activity that typically
occurred within 20 s of completion of dPAG microinjection. Respiration continued to increase until it reached a steady-state 3-5 min postinjection (see Fig. 2). The onset of the respiratory response was slower than for electrical stimulation but was sustained for the entire 520-s postinjection measurement period. As illustrated in Fig. 5, disinhibition of the dPAG was
associated with a dose-dependent decrease in both TI and
TE. TI and TE after 0.3 mM BicM
(n = 4) were significantly greater than after 0.5 mM
(n = 4), and that after 0.5 mM BicM was significantly
greater than that after 1.0 mM (n = 2) (Fig. 5).
Associated with the combined changes in TI and
TE, Ttot decreased and breathing frequency increased in a dose-dependent manner (Fig. 6).
Comparisons between control values and the 10-breath average taken
300 s after the offset of microinjection demonstrated significant
changes across all doses for TI, TE, Ttot, and
respiratory frequency relative to the preceding control (Table
2).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
Dose-related changes in TI (A) and
TE durations (B) after unilateral microinjection
of BicM into the dPAG. Zero reflects time that microinjection was
completed. Arrow indicates the position of the first average taken
immediately after microinjection completion. Values are means ± SE. * Significant differences between groups, P < 0.05. Statistical comparisons between doses at single 10-breath average
points after BicM administration were not indicated. Data from
animals receiving 0.3 and 0.5 mM BicM reflect average from 4 each. Data
from animals receiving 1 mM BicM reflect an average of 2.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Dose-related changes in Ttot (A) and
respiratory frequency (B) after unilateral microinjection of
BicM into the dPAG. Zero reflects time that microinjection was
completed. Arrow indicates the position of the first average taken
immediately after microinjection completion. See Fig. 5 for symbol
legend. Values are means ± SE. * Significant differences
between groups, P < 0.05. Statistical comparisons
between doses at single 10-breath average points after BicM
administration were not indicated. Data from animals receiving 0.3 and
0.5 mM BicM reflect average from 4 each.
|
|
BicM microinjection into the dPAG also elicited a marked increase in
baseline dEMG activity (Fig. 2). As shown in Fig.
7A, baseline activity
increased significantly in a dose-dependent manner: 0.5 and 1.0 mM were
significantly greater than 0.3 mM; 1.0 mM was significantly greater
than 0.5 mM. The average increase in baseline activity measured
300 s after the offset of microinjection had increased above
control level to a steady state of 611 ± 418% for 0.3 mM, over
733 ± 191% for 0.5 mM, and over 1,478 ± 741% for 1.0 mM.
The increase in baseline activity was sustained throughout the
postinjection measurement period (Fig. 7A). Comparisons
between the percent increase in baseline activity 300 s after the
offset of microinjection vs. the percent increase in baseline activity during electrical stimulation demonstrated no significant difference between baseline dEMG activity after 0.5 mM and electrical stimulation. The increase in baseline activity induced by 1.0 mM BicM, however, was
significantly greater, and 0.3 mM BicM was significantly less than
electrical stimulation. There was a significant increase in the delta
dEMG activity over control for all doses of BicM when measured 300 s after the offset of microinjection (133 ± 27% for 0.3 mM,
136 ± 26% for 0.5 mM, and 138 ± 4% for 1.0 mM). There
was, however, no significant difference in the delta dEMG activity
across doses (Fig. 7B). A comparison with the delta dEMG evoked during electrical stimulation demonstrated no significant difference across all three BicM doses.
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of increasing dose of BicM on baseline dEMG
activity and dEMG burst amplitude after unilateral microinjection
into the dPAG. Zero reflects time that microinjection was completed.
Arrow indicates the position of the first average taken immediately
after microinjection completion. See Fig. 5 for symbol legend. Values
are means ± SE. * Significant differences between groups,
P < 0.05. Statistical comparisons between doses taken
at single 10-breath average points after BicM administration were not
indicated. Data from animals receiving 0.3 and 0.5 mM BicM reflect
average from 4 each.
|
|
Associated with the significant increase in both delta dEMG and
respiratory frequency, there was a significant and dose-dependent increase in neural minute activity after BicM microinjection. The
change in neural minute activity was significantly less after 0.3 mM
than 0.5 and 1.0 mM BicM, whereas that after 0.5 mM was significantly
less than that after 1.0 mM (Fig. 8).
Ten-breath averages taken 300 s after the offset of microinjection
demonstrated that neural minute activity increased significantly above
control for all doses of BicM (205 ± 67% for 0.3 mM, 253 ± 68% for 0.5 mM, and 268 ± 2% for 1.0 mM). The increases in
neural minute activity above control associated with 0.5 mM BicM were
not significantly different from increases associated with electrical
stimulation. Increases in neural minute activity induced by 1.0 mM,
however, were significantly greater, and those induced by 0.3 mM were
significantly less, than electrical stimulation.
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 8.
Dose-related change in neural minute activity after
unilateral microinjection of BicM into the dPAG. Zero reflects time
that microinjection was completed. Arrow indicates the position of the
first average taken immediately after microinjection completion. Values
are means ± SE. * Significant differences between groups,
P < 0.05. Statistical comparisons between doses at
single 10-breath average points after BicM administration were not
indicated. Data from animals receiving 0.3 and 0.5 mM BicM reflect
average from 4 each.
|
|
Associated with the increase in respiration during disinhibition of the
dPAG, there was a simultaneous increase in both MAP and HR (Table 1).
The increase in MAP was dose related (14 ± 3 mmHg for 0.3 mM,
19 ± 6 mmHg for 0.5 mM, and 26 ± 12 mmHg for 1.0 mM). The
increase in HR induced by 1.0 mM BicM (34 ± 3 beats/min), however, was not significantly different from that induced by 0.5 mM
(65 ± 3 beats/min) or 0.3 mM (44 ± 8 beats/min)
BicM. Alternatively, the increase in HR induced by all three
doses of BicM was significantly greater than that induced by electrical
stimulation. Yet the increase in MAP induced by 0.3 mM BicM was not
significantly different from that induced by electrical stimulation.
The increase in MAP after 0.5 and 1.0 BicM was significantly greater
than that induced by electrical stimulation.
aCSF (n = 3) did not significantly change respiratory
(Fig. 9) or cardiovascular parameters
(Table 1) relative to control over the 3 min measured after central
microinjection into the dPAG.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of artificial cerebrospinal fluid microinjection
into the dPAG on TI, TE, and Ttot
(A) and neural minute activity (B). Zero reflects
time that microinjection was completed. Arrow indicates the position of
the first average taken immediately after microinjection completion.
Values are means ± SE from 3 animals.
|
|
| |
DISCUSSION |
The results of this study demonstrate for the first time that
disinhibition of neurons in the dPAG induces a profound increase in
respiratory muscle activity. The BicM-mediated disinhibition of dPAG
neurons is presumably via inhibition of GABAA receptors. This increase in respiratory muscle activity included changes in both
the Ttot and dEMG baseline activity and burst amplitude. The increase
in overall respiratory activity was accompanied by significant
increases in both MAP and HR. Whereas our results clearly demonstrated
that dPAG neurons involved in respiratory muscle activation receive
tonic inhibitory inputs, the central integration and descending
pathway(s) mediating this response remains unknown.
Breath-timing effects.
The effect of dPAG activation on breath timing resulted in an increase
in the rate of ventilation. Electrical stimulation elicited an
immediate increase in breathing frequency, with the first breath of the
stimulus-on condition exhibiting a decreased Ttot. The Ttot is divided
into inspiratory and expiratory durations. The overall decrease in Ttot
observed with electrical stimulation was the result of a decreased
TE with little change in TI. Bassal and Bianchi
(4) electrically stimulated the PAG without determination of the specific portion of the PAG activated. They also reported a
decrease in TE when single pulses were delivered during the expiratory phase.
The advantage of the electrical stimulation is the ability to observe
the latency of the stimulus-on and stimulus-off effects. The
stimulus-on response occurred within the first breath. The breathing
rate also rapidly returned to normal when the electrical stimulation
was removed. The first stimulus-off measurement period showed a
decrease in respiratory frequency to a level that was not significantly
different from the control period. In several animals, the first breath
after the stimulator was turned off showed a large inspiratory
activation indicative of a sigh or gasp. The TI and
TE increased, and the breathing frequency was similar to
prestimulation levels. These results suggest that there is a short
latency pathway between the dPAG and the neural centers mediating
breath timing. Increasing dPAG neuronal activity stimulates this timing
pathway, resulting in a change in the brain stem pattern generator.
The dPAG influence in breath timing is normally suppressed. Application
of BicM at the lowest dose in the present study resulted in an increase
in respiratory frequency greater than observed with electrical
stimulation. There was a dose-dependent increase in respiratory
frequency, with the highest dose eliciting more than a doubling in
respiratory frequency above resting levels to a rate of ~200
breaths/min. The disinhibition of dPAG increased frequency by
decreasing both TI and TE. The application of
BicM in all concentrations produced a progressive decrease in
TI and TE over the initial 6-10 breaths
and then remained at a steady-state level for hundreds of seconds. This
response was not observed with saline injections. Huang et al.
(18) also found an increased breathing frequency with
chemical stimulation of dPAG. Their increase in frequency was measured
as the 10-breath mean change in respiratory frequency during the
stimulation period. They reported a 50-60% increase in
respiratory frequency. The respiratory frequency change was a result of
a decrease in both TI and TE. The maximum
respiratory frequency reported in their study was 122 breaths/min with
60-nl DLH (3). In the present study, activation of the
dPAG with electrical stimulation increased respiratory frequency to
approximately the same rate, 120 breaths/min. Thus the disinhibition
effect has a greater effect on respiration than either electrical or chemical stimulation. Furthermore, the results of our study and those
of Huang et al. (18) suggest that either endogenous or chemical activation of the dPAG elicits a change in the respiratory control center, resulting in an increase in cycle rate and a decrease in cycle time by an effect on both inspiratory and expiratory durations. In contrast, electrical stimulation appears to predominantly modulate expiratory duration.
Diaphragm EMG effects.
The stimulation of dPAG neurons increased dEMG activity. The peak of
the integrated diaphragm signal increased in the first breath during
which electrical stimulation was applied. The peak dEMG was elevated by
an increase in both the magnitude of the inspiratory burst activity and
an increase in tonic baseline activity. Bassal and Bianchi
(4) also mentioned an augmentation of phrenic burst
activity with electrical stimulation of the PAG. Huang et al.
(18) noted, but did not analyze, an increase in diaphragm activity when DLH was injected into the dPAG. Examination of their Fig.
1 shows an increase in inspiratory burst amplitude and an apparent
increase in baseline activity with the higher dose of DLH. In the
present study, we were able to partition the increase in diaphragm
activity with dPAG stimulation into two components: change in activity
and baseline activity. While the magnitude of the inspiratory burst
(change in EMG) increased by ~50%, the baseline activity increased
4- to 10-fold. This suggests a dual effect on the magnitude of the
drive to the diaphragm, a large increase in resting muscle tone, and an
increased phasic activation. Although we did not measure respiratory
mechanical ventilation, the increased baseline tone suggests that
functional residual capacity (FRC) may have increased by less
relaxation during expiration. Future studies recording the mechanical
effect of this increased diaphragm activation are needed.
Disinhibition of the dPAG again had a greater effect on the dEMG
activity than electrical stimulation. There was a dose-dependent increase in peak integrated dEMG activity primarily due to a
dose-dependent progressive increase in baseline dEMG activity. The
inspiratory burst amplitude (delta dEMG) increased for all three doses,
but there was no significant difference in either the time course of
the response or the strength of the response as a function of dose. The
change in diaphragm activity reached a plateau at ~30-40% above
normal. The baseline activity, however, increased in a dose-dependent
manner. The increase ranged from 2- to 15-fold. This indicates that
there was a profound increase in phrenic motor neuron activity during
the expiratory phase, again suggesting reduced diaphragm relaxation
during expiration and an increase in FRC. The specific neural pathways
mediating this response are unknown. It is likely that the dPAG
increases the cycle timing and inspiratory magnitude of the brain stem
neural oscillator. It is also possible that the descending influence of
the dPAG acts in parallel to the respiratory oscillator acting on
phrenic motor neurons to increase their steady-state excitability, thus producing an increase in the baseline diaphragm EMG.
The combined ventilatory drive response to dPAG activation by direct
stimulation or disinhibition is illustrated by the change in neural
minute activity. Similar to previous reports using phrenic integrated
electroneurogram magnitude and cycle rate (14), we estimated the ventilatory drive by multiplying the respiratory rate
times the magnitude of the inspiratory dEMG burst (change in
activity). Although this is not a measure of minute
ventilation, it does allow us to predict (14) the
ventilatory effect of dPAG activation. Both direct activation by
electrical stimulation and disinhibition elicited an increase in the
neural minute activity. With disinhibition, the dose-dependent effect
was due to the dose-dependent change in respiratory frequency because
there was no dose-dependent effect on inspiratory burst magnitude. The
results indicate that the greater the degree of activation of the dPAG,
the greater the increase in ventilation. The increased neural minute
activity occurs in the presence of an increased tonic EMG activation.
Future studies are needed to determine whether the increase in neural ventilation results in an increase in mechanical ventilation. It is
possible that mechanical minute ventilation is not increased because of
the possible increase in FRC due to the increase in baseline dEMG activity.
Cardiovascular effects.
Both chemical and electrical stimulation of the dPAG induced
significant increases in MAP and HR. The cardiovascular changes that we
observed were similar to those reported by other investigators in both
conscious and anesthetized preparations (10, 13, 27, 29).
The increases in MAP and HR were proportional to the level of dPAG
activation. Furthermore, comparisons between electrical vs. chemical
stimulation suggest that disinhibition of the dPAG, particularly after
0.5 and 1.0 mM BicM, had a significantly greater effect on sympathetic
drive compared with electrical stimulation. Recruitment of
sympathoexcitatory neurons in the rostral ventrolateral medulla has
been shown to mediate dPAG-evoked pressor and tachycardia responses
(20, 33). DPAG activation has also been documented to
attenuate baroreflex function (27, 30). Although
baroreflex function was not assessed in the present study, it is
possible that a portion of dPAG-evoked change in respiration may have
been secondary to altered baroreflex function; blockade of baroreceptor input to the brain stem increases respiratory drive through changes in
both TI and TE (17).
Summary.
Simultaneous increases in MAP, HR, and respiration, coupled with
attenuation of baroreflex function, prepare the animal to meet the
physiological demands of the body during "flight or fight" responses (1). The dPAG and nearby structures have been
identified as one of the few regions of the brain to contain the neural
components sufficient to coordinate these behaviors (2, 11, 29,
30, 34). The presence of tonic inhibitory input to the dPAG has been well documented in relation to both behavioral and cardiovascular components of the defense response (6, 8, 29, 31). Similar to other regions of the brain, including certain respiratory areas (24, 26), tonic suppression and the subsequent withdrawal of inhibition allow for rapid gain adjustment of excitatory inputs (36). Although the exact origin of this inhibitory input
is unknown, descending projections from forebrain structures as well as
local GABAergic interneurons may contribute to tonic suppression of
dPAG neurons (1, 7, 16). In the present study, we used BicM to block this tonic inhibitory input. In addition to selectively blocking GABAA-receptor function, BicM has been shown to
block Ca2+-activated K+ currents. Blockade of
these K+ currents increases neuronal discharge rate,
independent of GABAA-receptor blockade. We did not test the
effects of a second receptor antagonist; thus we cannot completely rule
out confounding effects of altered K+ conductances. Yet
results from other investigators suggest that our findings were
primarily due to the withdrawal of tonic inhibitory inputs. For
example, microinjection of the noncompetitive
GABAA-receptor blocker picorotoxin produces a similar
increase in MAP, HR, and respiratory frequency as that described after
BicM microinjection (29). Furthermore, both in vitro and
in vivo, GABAA-receptor blockade increases synaptic
activity and blocks the inhibitory effect of GABA (22,
29). In vitro, the influence of BicM on neuronal discharge is
blocked after synaptic blockade (29). Thus we are
reasonably confident that the changes in respiratory cycle activity
that we report here are primarily the result of neuronal disinhibition
and reflect the expression of another component of the behavioral
defense response.
In conclusion, the results of the present study demonstrate that
neurons of the dPAG are involved in modulating respiratory function and
are normally suppressed by GABAA inhibition. Furthermore, tonic excitatory inputs to these neurons can be uncovered by
GABAA-receptor blockade. Disinhibition of the dPAG produces
a powerful increase in respiratory activity characterized by a decrease
in Ttot, increased inspiratory burst amplitude, and an increase in
diaphragm activity during expiration. The pathways mediating these
changes remain to be determined.
| |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grant HL-63232.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
P. W. Davenport, Dept. of Physiological Sciences, College of
Veterinary Medicine, PO Box 100144, HSC, Univ. of Florida, Gainesville,
FL 32610-0144 (E-mail:
davenportp{at}mail.vetmed.ufl.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.
First published November 15, 2002;10.1152/japplphysiol.00740.2002
Received 9 August 2002; accepted in final form 4 November 2002.
| |
REFERENCES |
1.
Bandler, R,
Keay KA,
Floyd N,
and
Price J.
Central circuits mediating patterned autonomic activity during active vs. passive emotional coping.
Brain Res Bull
53:
95-104,
2000[Web of Science][Medline].
2.
Bandler, R,
Prineas S,
and
McCulloch B.
Further localization of midbrain neurons mediating the defense reaction in the cat by microinjections of excitatory amino acids.
Neurosci Lett
56:
311-316,
1985[Web of Science][Medline].
3.
Bandler, R,
and
Shipley MT.
Columnar organization in the midbrain periaqueductal gray: modules for emotional expression?
Trends Neurosci
17:
379-389,
1994[Web of Science][Medline].
4.
Bassal, M,
and
Bianchi AL.
Inspiratory onset or termination induced by electrical stimulation of the brain.
Respir Physiol
50:
23-40,
1982[Web of Science][Medline].
5.
Behbehani, M.
Functional characteristics of the midbrain periaqueductal gray.
Prog Neurobiol
46:
575-605,
1995[Web of Science][Medline].
6.
Behbehani, MM,
Jiang M,
Chandler SD,
and
Ennis M.
The effect of GABA and its antagonists on midbrain periaqueductal gray neurons in the rat.
Pain
40:
195-204,
1990[Web of Science][Medline].
7.
Bernard, J,
and
Bernard R.
Parallel circuits for emotional coping behavior: new pieces in the puzzle.
J Comp Neurol
401:
429-436,
2000.
8.
Brandao, M,
Anseloni VZ,
Pandossio JE,
De Araujo JE,
and
Castilho VM.
Neurochemical mechanisms of defensive behavior in the dorsal midbrain.
Neurosci Biobehav Rev
23:
863-875,
1999[Web of Science][Medline].
9.
Brannan, S,
Liotti M,
Egan G,
Shade R,
Madden L,
Robillard R,
Abplanalp B,
Sofer K,
Denton D,
and
Fox PT.
Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air.
Proc Natl Acad Sci USA
98:
2029-2034,
2001[Abstract/Free Full Text].
10.
Carobrez, AP,
Schenberg LC,
and
Graeff FG.
Neuroeffector mechanisms of the defense reaction in the rat.
Physiol Behav
31:
439-444,
1983[Medline].
11.
Carrive, P,
and
Bandler R.
Control of extracranial and hindlimb bloodflow by the periaqueductal gray of the cat.
Exp Brain Res
84:
599-606,
1991[Web of Science][Medline].
12.
Carrive, P,
and
Bandler R.
Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal gray: a correlative functional and anatomical study.
Brain Res
541:
206-215,
1991[Web of Science][Medline].
13.
Carrive, P,
Bandler R,
and
Dampney RAL
Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray.
Brain Res
493:
385-390,
1989[Web of Science][Medline].
14.
Eldridge, FL.
Relationship between respiratory nerve and muscle activity and muscle force output.
J Appl Physiol
39:
567-574,
1975[Abstract/Free Full Text].
15.
Fendt, M.
Differential regions of the periaqueductal gray are involved in the expression and conditioned inhibition of fear-potentiated startle.
Eur J Neurosci
10:
3876-3884,
1998[Web of Science][Medline].
16.
Griffiths, JL,
and
Lovick TA.
Co-localization of 5HT-2A-receptor- and GABA-immunoreactivity in neurons in the periaqueductal gray matter of the rat.
Neurosci Lett
326:
151-154,
2002[Web of Science][Medline].
17.
Hopp, F,
and
Seagard JL.
Respiratory responses to selective blockade of carotid sinus baroreceptors in dogs.
Am J Physiol Regul Integr Comp Physiol
275:
R10-R18,
1998[Abstract/Free Full Text].
18.
Huang, ZG,
Subramanian SH,
Balnave RJ,
Turman AB,
and
Chow CM.
Roles of periaqueductal gray and nucleus tractus solitarius in cardiorespiratory function in the rat brainstem.
Respir Physiol
120:
185-195,
2000[Web of Science][Medline].
19.
Jurgens, U,
and
Lu CL.
The effects of periaqueductally injected transmitter antagonist forebrain-elicited vocalization in the squirrel monkey.
Eur J Neurosci
5:
735-741,
1993[Web of Science][Medline].
20.
Li, P,
and
Lovick TA.
Excitatory projections from hypothalamic and midbrain defense regions to nucleus paragiagantocellularis lateralis in the rat.
Exp Neurol
89:
543-553,
1985[Web of Science][Medline].
21.
Lovick, T.
Inhibitory modulation of cardiovascular defense response by the ventrolateral periaqueductal gray matter in rats.
Exp Brain Res
89:
133-139,
1992[Web of Science][Medline].
22.
Lovick, T.
Involvement of GABA in medullary raphe-evoked modulation of neuronal activity in the periaqueductal gray matter in the rat.
Exp Brain Res
137:
214-218,
2001[Web of Science][Medline].
23.
Lui, K,
Solomon NP,
and
Luschei ES.
Midbrain regions for eliciting vocalization by electrical stimulation in anesthetized dogs.
Ann Otol Rhinol Laryngol
107:
977-986,
1998[Web of Science][Medline].
24.
McCrimmon, D,
Zuperku EJ,
Hayashi F,
Dogas Z,
Hinrichsen CFL,
Stuth EA,
Tonkovic-Capin M,
Krolo M,
and
Hopp FA.
Modulation of synaptic drive to respiratory premotor and motoneurons.
Respir Physiol
110:
161-176,
1997[Web of Science][Medline].
25.
Nakai, M,
and
Maeda M.
Systemic and regional haemodynamic responses elicited by microinjection of N-methyl-D-aspartate into the lateral periaqueductal gray matter in anesthetized rats.
Neuroscience
58:
777-783,
1994[Web of Science][Medline].
26.
Nattie, E,
Shi J,
and
Li A.
Bicuculline dialysis in the retrotrapezoid nuclues (RTN) region stimulates breathing in the awake rat.
Respir Physiol
124:
179-193,
2001[Web of Science][Medline].
27.
Nosaka, S,
Murata K,
Inui K,
and
Murase S.
Arterial baroreflex inhibition by midbrain periaqueductal gray in anesthetized rats.
Pflügers Arch
424:
266-275,
1993[Web of Science][Medline].
28.
Paxinos, G,
and
Watson C.
The Rat Brain in Stereotaxic Coordinates. New York: Academic, 1998.
29.
Schenberg, LC,
De Aguiar JC,
and
Graeff FG.
GABA modulation of the defense response induced by electrical brain stimulation.
Physiol Behav
31:
429-437,
1983[Medline].
30.
Schenberg, LC,
Vasquez EC,
and
da Costa MB.
Cardiac baroreflex dynamics during the defense reaction in freely moving rats.
Brain Res
621:
50-58,
1993[Web of Science][Medline].
31.
Schmitt, P,
Carrive P,
Di Scala G,
Jenck F,
Brandao M,
Bagri A,
Moreau JL,
and
Sandner G.
A neuropharmacological study of the periventricular neural substrate involved in flight.
Behav Brain Res
22:
181-190,
1986[Web of Science][Medline].
32.
Shaikh, M,
and
Siegel A.
GABA-mediated regulation of feline aggression elicited from midbrain periaqueductal gray.
Brain Res
507:
51-56,
1990[Web of Science][Medline].
33.
Verberne, AJ,
and
Guyenet PG.
Midbrain central gray: influence on medullary sympathoexcitatory neurons and the baroreflex in rats.
Am J Physiol Regul Integr Comp Physiol
263:
R24-R33,
1992[Abstract/Free Full Text].
34.
Verberne, AJ,
and
Struyker Boudier HA.
Midbrain central grey: regional haemodynamic control and excitatory amino acidergic mechanisms.
Brain Res
550:
86-94,
1991[Web of Science][Medline].
35.
Zhang, S,
Davis PJ,
Bandler R,
and
Carrive P.
Brainstem integration of vocalization: role of the midbrain periaqueductal gray.
J Neurophysiol
72:
1337-1356,
1994[Abstract/Free Full Text].
36.
Zuperku, E,
and
McCrimmon DR.
Gain modulation of respiratory neurons.
Respir Physiol
131:
121-133,
2002.
J APPL PHYSIOL 94(3):913-922
8750-7587/03 $5.00
Copyright © 2003 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
