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Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Cream, Carlos L., Aihua Li, and Eugene E. Nattie. RTN
TRH causes prolonged respiratory stimulation. J. Appl.
Physiol. 83(3): 792-799, 1997.
We injected
thyrotropin-releasing hormone (TRH; 10 nl; 0.25, 0.5, 1.0, or 10 mM),
its inactive free acid form (TRHOH; 1 mM), or a metabolite with low
TRH-receptor binding affinity, histidine-proline diketopiperazine (cHP;
1 mM), into the retrotrapezoid nucleus of anesthetized rats. Injection
location was verified by anatomic analysis. Lower doses (0.25-0.5
mM) significantly increased both the product of integrated phrenic
amplitude and frequency
(
Phr · f) and f for 20-30
min compared with artificial cerebrospinal fluid control injections. Higher doses (1.0-10 mM) produced greater and long-lasting
stimulation of Phr · f,
Phr, and f and of blood pressure. This
stimulation reached values 150% of baseline and durations of 270 min
after a single injection. TRHOH (1 mM ) or cHP (1 mM) had no effect on
Phr but increased f, as did 1 mM TRH. We conclude
that TRH has a very powerful stimulatory effect in the retrotrapezoid
nucleus region on Phr · f, with
the Phr response seemingly specific for TRH
receptors. Similar responses of f to TRHOH and cHP suggest it may be
nonspecific.
control of breathing; ventrolateral medulla; thyrotropin-releasing
hormone free acid; histidine-proline diketopiperazine; blood pressure; respiratory regulation; retrotrapezoid nucleus
INTRODUCTION THE ROSTRAL VENTROLATERAL MEDULLA (RVLM) contains
neurons that are important in the control of breathing. Chemoreception
occurs there at topographically defined areas within and rostral to, as
well as caudal to, the RVLM (5). Cooling of the ventral medullary
surface at a topographical location lying between these chemosensitive
areas (the intermediate area) causes a significant depression of
CO2 sensitivity and ventilation,
often to apnea, in anesthetized animals (1, 9, 20), with similar but
less marked effects in unanesthetized preparations (1, 9).
An important component of the RVLM is the retrotrapezoid nucleus (RTN),
a set of superficial neurons near the surface of the RVLM that lies
ventral and ventromedial to the facial nucleus at the topographical
border of the rostral chemosensitive zone and the intermediate area of
the ventral medullary surface. The RTN connects with both the dorsal
and ventral respiratory groups (26) and contains neurons that fire in
synchrony with the respiratory rhythm (22). Chemical lesions of the RTN
in anesthetized animals result in decreased phrenic nerve activity
(PNA), often resulting in apnea, and loss of responsiveness to systemic
CO2 (20). In unanesthetized rats,
unilateral lesions affecting ~35% of the RTN neurons reduce
CO2 sensitivity by 39% but have
no effect on baseline ventilation (1). Microinjection of ionotropic
glutamate-receptor antagonists into the RTN of anesthetized cats
results in decreased PNA and loss of responsiveness to
CO2 (19); injection of glutamate results in an increase in PNA (16). Injection of metabotropic glutamate-receptor antagonists have little effect on baseline PNA, but
injection of metabotropic agonists results in a prolonged increase in
PNA sustained for >45 min (16). Muscarinic antagonists also diminish
PNA and the response to increased systemic
CO2 (21).
In this study we ask whether injection of thyrotropin-releasing hormone
(TRH;
L-pyroglutamyl-L-histidyl-L-prolinamide)
into the RTN region of anesthetized rats has any effect on breathing as
measured by the product of integrated phrenic amplitude and frequency
We hypothesized that unilateral RTN injections of TRH in artificial
cerebrospinal fluid (aCSF) would stimulate our measurement of
ventilatory output, i.e.,
METHODS
Phr · f). The rationale for
this study is as follows. 1)
Injection of substance P into the RTN region stimulates respiratory
output (3). 2) Substance P-like
immunoreactivity colocalizes with TRH-like immunoreactivity in neurons
near the RTN region (14, 25). 3)
Neurons in the midline raphe and parapyramidal areas nearby to the RTN
region contain TRH-like immunoreactivity (25) and have known
projections into the RTN region (30).
4) Although no specific observations
demonstrate TRH receptors in the RTN region, existing data for rat
brain stem show TRH receptors in this general vicinity (17).
5) Administration of TRH via the cerebral ventricles (12, 13, 28) or by microinjection at midline
ventral medullary sites caudal to the RTN region (18) stimulates
respiratory output. Microinjection of TRH into the more dorsally
located nucleus tractus solitarii (NTS) also results in an increase in
ventilatory output (12, 18).
Phr · f. Doses of 0.25 and 0.5 mM TRH increased f and
Phr · f; 1.0 and 10 mM doses
increased f, Phr, and
Phr · f. Control injections of
equal volumes of aCSF had no effect, whereas injections of the inactive TRH free acid form (TRHOH; 1 mM in aCSF) and injection of the TRH
metabolite (cHP; histidine-proline diketopiperazine), which has
essentially no binding to the TRH receptor in vitro (11, 17), increased
f.
Phr, f, total cycle duration, the
duration of inspiration (TI)
and expiration, and mean arterial blood pressure were computed on line
by using locally written software. We also computed the neural
equivalent of minute ventilation, i.e.,
Phr · f.
After surgery, the animal was allowed to stabilize for 20-30 min.
A CO2 response curve was
determined by increasing the inspired fraction of
CO2 and monitoring the
Phr and f measured at end-tidal
CO2 values of 5, 7, and 9%. The
Phr at 9% end-tidal
CO2 was defined as the maximum or
100%. CO2 was then lowered back
to an end-tidal value of 4-5% for baseline measurements. All of
the animals had baseline Phr values between 45 and 60% of maximum. Subsequently, for comparisons among animals, all
Phr · f and
Phr values are expressed as a percentage of
baseline.
Microinjections (10 nl) were made with a Pico-spritzer (General Valve
II) over a 3-s period. All drugs were dissolved in aCSF and
equilibrated with CO2 to ~pH
7.4. The composition of the aCSF (in mM) was 152 sodium, 3.0 potassium,
2.1 magnesium, 2.2 calcium, 26 chloride, and 25 bicarbonate. The
calcium was added after the aCSF was warmed to 37°C and
equilibrated with 5% CO2. For
most injections, the movement of the meniscus in the tubing leading to
the micropipette was monitored as an index of injection volume. To
allow postmortem evaluation of the injection site and determination of
the volume injected, fluorescein or rhodamine latex beads
(Polysciences) were added to each injection. After the injection,
phrenic and blood pressure measurements were taken at 10-min intervals
until Phr returned to baseline.
At the end of the experiment, animals were killed by intravenous
infusion of saturated KCl. The brain was rapidly removed and the
medulla was dissected and frozen. It was then sectioned at 20 µm in a cryostat. Injection sites were identified by
using a fluorescence microscope to localize the center of each
injection, and serial sections were stained with cresyl violet to
identify anatomic landmarks. The volume of each injection was estimated anatomically by a geometric approach, by which we first measured the
total rostral-to-caudal length of the region of fluorescence and the
largest cross-sectional area among the serial sections and then
calculated volume by using the formula for adjoining cones (1). The
center of each injection was also located in millimeters caudal to the
bregma.
At each measurement time during the protocol, we assessed the mean
value for all variables over a period of 10 respiratory cycles. In
preliminary experiments, it was apparent that lower TRH doses produced
short-lasting responses, whereas higher doses produced longer duration
responses. Accordingly, we used two control groups. The first
procedure consisted of four injections of aCSF into the
RTN in four animals that were part of the protocols for low-dose TRH,
TRHOH, or cHP injections, all groups in which data were collected at
10, 20, and 30 min after the injection. The second control group
consisted of six animals that received an aCSF injection of 10 nl into
the RTN with measurements made at 10, 20, 30, 60, 90, and 120 min.
Responses to 1.0 and 10 mM TRH were compared with this separate control
group. Many of these TRH responses actually lasted for much longer time
periods, but the formal statistical analysis is constrained to this 2-h
period. These controls were used in a prior study (20) and are included here because the experiments were performed in the same time period and
under the same experimental conditions as these TRH injections.
For TRH injections, an increase in
Phr · f that was greater than
that observed after any control injection was classified as a response.
In practice, this was an increase in
Phr · f that
1) was greater than a 10% change in
Phr · f expressed as a
percentage of the maximum and 2) was
present at least over 20 min. For mean arterial blood pressure, a
response was a >12-mmHg increase sustained over at least 20 min. For
these TRH injections, we report only the responders as defined above.
Mean values of Phr · f,
Phr, f, and mean arterial blood pressure for each experimental group at each time period were compared by performing a
two-way analysis of variance (Systat) to examine treatment and time
with Tukey's post hoc analysis as appropriate. The first comparison
was among the short time period 30-min aCSF controls and the responders
to 0.25 and 0.5 mM TRH; the second comparison was among the 120-min
aCSF controls and the responders to 1 and 10 mM TRH. The final analysis
compared all TRHOH and cHP injections (each of 1 mM concentration) with
the responders to 1 mM TRH.
RESULTS
There were 43 injections made into the RTN region in 28 rats as
determined by postmortem anatomic analysis. These animals maintained a
mean arterial blood pressure at 90 mmHg or greater for the duration of
the experiments and demonstrated a good initial response to increased
CO2, i.e., a doubling or greater
of Phr · f. This indicates a
healthy ventral medulla in this type of preparation.
In the low-dose range, four TRH injections (2 with 0.25 mM; 2 with 0.5 mM) produced a significant increase in
Phr · f
(P < 0.003) and in f
(P < 0.02) compared with four aCSF
control injections (Fig. 1). In this, and
every other case in this study, the increase in f resulted from a
shortening of TI. Responses to
four TRH injections (1 with 0.25 mM; 3 with 0.5 mM) are
indistinguishable from the control data shown in Fig. 1. Figure
2 shows the anatomic location of the center
of each of the eight injections shown in Fig. 1. The center of the four
control injections was 10.75 ± 0.16 (SE) mm caudal to bregma and
that of the four TRH injections was 10.89 ± 0.25 mm; the control
injection volume calculated from the fluorescent bead distribution was
9.1 ± 2.6 nl; that of the TRH injections was 21 ± 6.1 nl. Mean
arterial blood pressure was unchanged. The locations of the 0.25 and
0.5 mM TRH injections that did not produce a response were also within
the RTN region (data not shown).
Phr · f; neural index of
minute ventilation; A),
Phr (B), and f
(C) as a function of time. Values
are means ± SE. 
, Values for 4 control artificial cerebrospinal
fluid (aCSF) injections; 
, values for 4 injections of
thyrotropin-releasing hormone (TRH) at 0.25 mM (2) and 0.5 mM (2)
concentrations that produced responses. Expressed as %baseline, both
Phr · f and f were
significantly increased [P < 0.003 for Phr · f and
P < 0.03 for f; 2-way analysis of
variance (ANOVA)]. Values for 4 nonresponders receiving TRH at
these doses are indistinguishable from control values and are not
shown.
RTN injection of 1 mM (n = 9) and 10 mM (n = 3) TRH produced responses in
every case. Figure 3 shows an example of a
1 mM TRH injection. Phr · f is
increased within 10 min to a value of 71% of baseline, reaching a peak
value of 150% of baseline at 60 min, then slowly declining to a value
of 33% of baseline at 270 min. Both Phr and f
show similar responses, as does mean arterial blood pressure. The
injection site is in the RTN region, 11.06 mm caudal to bregma, and the
calculated injection volume is 20 nl. Of the nine 1 mM TRH injections,
Phr · f returned to baseline or
the experiment was stopped at 60 (n = 2), 120 (n = 3), 180 (n = 2), and 270 (n = 2) min. Of the three 10 mM TRH
injections, Phr · f returned to
baseline, or the experiment was stopped at 120, 240, and 270 min.
Phr · f (;
A), Phr (;
B), f (
;
C), and mean arterial blood pressure
(MAP; 
; D) after injection of TRH
at 1 mM concentration into retrotrapezoid nucleus (RTN) region. Data are expressed as %baseline. Bottom
left, exact location of center of injection with
pattern of fluorescent beads inside small square. Bar, 1 mm.
With such long-duration effects, we focused the statistical analysis on
the first 120 min by using a control group that received only 10 nl
aCSF into the RTN and had measurements made over this time period.
These data are shown in Fig. 4.
Phr · f was increased
significantly compared with control by the 1 mM
(P < 0.001) and 10 mM TRH injections
(P < 0.001), and these two TRH
injection responses differed from each other
(P < 0.001). Phr was increased significantly compared with
control by the 1 mM (P < 0.001) and
10 mM TRH injections (P < 0.001).
These two TRH injection responses did not differ from each other.
Compared with control, f was increased significantly by the 1 mM
(P < 0.03) and 10 mM TRH injections
(P < 0.001), and these two TRH
injection responses differed from each other
(P < 0.001). Figure
5 shows the anatomic location of the center
of each injection summarized in Fig. 4. The center of the six control injections was 11.22 ± 0.12 mm caudal to bregma, that of the nine 1 mM TRH injections, 10.96 ± 0.07 mm, and that of the three 10 mM TRH
injections, 11.1 ± 0.29 mm; the control injection volume calculated
from the fluorescent bead distribution was 17.4 ± 4.6 nl; that of
the nine 1 mM TRH injections, 9.2 ± 1.6 nl; and that of the three
10 mM TRH injections, 23.7 ± 4 nl.
;
n = 6), 1 mM TRH injections that
produced responses (; n = 9), and
10 mM RTN TRH injections that produced responses (;
n = 3) are shown for
Phr · f
(A), Phr
(B), and f
(C) as a function of time. Values
are means ± SE and are expressed as %baseline. Note interrupted
time scale. For Phr · f,
responses of both 1 and 10 mM doses are significantly different from
control (P < 0.001; 2-way ANOVA),
and responses of these 2 doses differ from each other
(P < 0.001; 2-way ANOVA; Tukey's post hoc test). For Phr, responses of both 1 and
10 mM doses are significantly different from control (P < 0.001; 2-way ANOVA), and
responses of these 2 doses do not differ from each other
(P < 0.001; 2-way ANOVA; Tukey's
post hoc test). For f, responses of both 1 and 10 mM doses are
significantly different from control
(P < 0.03;
P < 0.001; 2-way ANOVA), and responses of these 2 doses differ from each other
(P < 0.001; 2-way ANOVA; Tukey's
post hoc test).
Mean arterial blood pressure was increased in four of nine 1 mM TRH
injections and two of three 10 mM TRH injections. The change in mean
arterial blood pressure for these six responses in shown on Fig.
6 along with the mean change in blood
pressure for the six control animals that received RTN injections of
aCSF. All six of these RTN TRH injections with increased blood pressure also had a response in terms of increased
Phr · f. Their injection
locations are among those shown on Fig. 5 and did not differ from the
locations of injections that had no associated phrenic or blood
pressure response as far as we could tell from our anatomic analysis.
The center of the injections that affected blood pressure was 10.93 ± 0.07 mm caudal to bregma; the injection volume calculated from
the fluorescent bead distribution was 12.6 ± 2.9 nl.
) in MAP for 6 control aCSF injections into RTN () and
for 6 injections of TRH at 1 mM (n = 4) and 10 mM (n = 2) concentrations
into RTN as a function of time. Values are means ± SE.
Responses to five injections of TRHOH at 1 mM concentration are shown
in Fig. 7 compared with the responses of
the nine 1 mM TRH injections.
Phr · f is significantly lower
after TRHOH injection (P < 0.03) as
is Phr (P < 0.03), but the response in terms of frequency is similar after each
type of injection. The location of these TRHOH injections is shown
(Fig. 7, bottom left). The center of
the TRHOH injections was 11.0 ± 0.23 mm caudal to bregma; the
injection volume calculated from the fluorescent bead distribution was
9.7 ± 2.8 nl. None of these injections increased mean arterial
blood pressure.
Phr · f
(A), Phr
(B), and f
(C) for initial 30 min of response to 1 mM TRH injected into RTN () and for 5 injections into RTN of 1 mM TRHOH (), TRH free acid with very little affinity for TRH
receptor (see text). Values are means ± SE.
Phr · f and
Phr responses differ significantly between TRH
and TRHOH (P < 0.02; 2-way ANOVA),
whereas f responses do not differ. Bottom
left, center of 5 TRHOH injections as described for
Figs. 2 and 5.
Responses to six injections of the TRH metabolite cHP at 1 mM
concentration are shown in Fig. 8 compared,
as for TRHOH injections, with the 1 mM TRH responses.
Phr · f and
Phr are significantly lower than after equimolar
TRH injection (P < 0.01), and the
change in f is very similar. The cHP injection locations are shown in
Fig. 8. The center of the injections was 10.51 ± 0.13 mm caudal to bregma; the injection volume calculated from the fluorescent bead distribution was 7.9 ± 1.8 nl.
Phr · f
(A), Phr
(B), and f
(C) for initial 30 min of response to 1 mM TRH injected into RTN () and for 6 injections into RTN of 1 mM histidine-proline diketopiperazine (cHP; ), a TRH metabolite with
very little affinity for TRH receptor (see text). Values are means ± SE. Phr · f and
Phr responses differ significantly between TRH
and cHP (P < 0.01; 2-way ANOVA),
whereas f responses do not differ. Bottom
left, center of 6 cHP injections as described for Figs.
2 and 5.
DISCUSSION
Phr · f
in a dose-dependent manner. With doses in the 0.25 to 0.5 mM range, the
effect is on f, lasts 20-30 min, and is not associated with
changes in blood pressure. With doses in the 1 to 10 mM range the
effect is large, up to a 150% increase in
Phr · f, includes increases in
Phr and f, is of long duration, up to 270 min or
more and, in some cases, is associated with a similarly long-lasting
increase in blood pressure.
Our effective TRH dose range is very similar to that described in
experiments with intracerebroventricular injection of TRH in conscious
rats (28). We have no evidence as to the actual concentration of TRH
released naturally in vivo in the synaptic cleft. In general, it is an
interesting feature that in microinjection studies performed in vivo,
as was ours, the concentrations needed for physiological effect are
larger than those needed for effects in vitro or for binding to
isolated receptors. For TRH, in vitro slice experiments use
concentrations in the micromolar range (2, 6, 8, 23, 24), and
membrane-binding studies use concentrations in the nanomolar range
(11), whereas we used concentrations in the millimolar range. It is
often assumed that the low concentrations effective in vitro represent
the normal physiology but, for glutamate, estimates of the
concentration in the synaptic cleft in vivo reach the millimolar range
(4). For TRH, the absence of an effective antagonist makes it difficult
to conclude which in vivo TRH concentrations reflect an event that
could mirror normal physiology.
Controls.
Control injections of aCSF had very little effect on
Phr · f,
Phr, or f over either 30 or 120 min. Separate
controls used TRHOH, the free acid, with a 1,000-fold lower affinity
for TRH receptors in vitro (11, 17), and a TRH metabolite, cHP. At the
dose level of 1 mM, part of the increased f response to TRH could be
attributable to a nonspecific effect because the free acid produced a
similar effect on f. cHP, with virtually no affinity for TRH receptors (11), also increased f, similarly suggesting an effect that is not
specific for TRH receptors. The large f response to RTN TRH injections
may represent a mechanism different from that of the
Phr response and unrelated to specific stimulation of TRH receptors.
Technical issues.
TRH can counteract the sedative effects of anesthetic agents (15); it
has a net excitatory effect on the central nervous system, perhaps via
the reticular formation. We monitored the level of anesthesia by using
blood pressure, respiratory rate, and responses of these variables to a
pinch of the footpad or tail. In these TRH experiments we monitored
pain-induced responses more frequently and gave additional doses of
anesthesia more liberally than we normally do. This possible
interaction of TRH with anesthesia depth is a difficult issue given the
long-lasting nature of the TRH effects after the 1 and 10 mM doses. We
believe that the meticulous attention paid to the depth of anesthesia
makes it unlikely that the large and long-duration TRH effects can be
attributable to changes in the depth of anesthesia. Experiments in
conscious rats with the intracerebroventricular injection of TRH at 0.5 to 5 mM concentrations support this interpretation. In these
experiments, ventilation, tidal volume, and f were increased by very
large amounts for the 60 min during which they were recorded, with no evidence of a waning response at the end of the 60 min (28).
In monitoring the 10-nl injection volume, in addition to measuring, in
some experiments, the movement of the meniscus at the air-liquid
interface within the tubing to the micropipette, we evaluated the size
of the injections by postmortem measurement of the distribution volume
of the injected fluorescent beads. For all 43 injections, the volume
estimated by this analysis was 12.8 ± 1.3 nl. This volume did vary
among the experimental groups, but the variability was small, with the
cHP group having the smallest mean injection volume, 7.9 ± 1.8 nl,
and the 10 mM TRH group having the largest mean injection volume, 23.7 ± 4 nl. The actual volume of distribution of the TRH is most likely
slightly larger than the injected volume because it will diffuse to a
slightly larger region, the degree of this diffusion being determined,
in part, by the diffusion coefficient of the injected substance. Of
interest here are measurements by autoradiography of the volume of
distribution of radiolabeled TRH injected at 50-, 100-, or 200-nl
amounts into the hypothalamic region (29). The 50-nl injected volume
had a 77% recovery within the localized injection site; the larger injection volumes showed much greater spread into surrounding tissue.
Our much smaller injections most likely have an even more circumscribed
effective region. The center of the 43 injections reported in this
study were well within the RTN region.
The RTN.
The RTN region was defined initially by retrograde tracing studies
after injection into the ventral or dorsal respiratory group (26). In
the rat (22) this region lies, at its most caudal point, at the level
of the rostral aspect of the retrofacial nucleus ventrolateral to the
nucleus paragigantocellularis lateralis 100-300 µm dorsal to the
ventral medullary surface. It continues in the rostral direction, lying
ventral and ventromedial to the facial nucleus. Identified by such
retrograde tracing studies, this nucleus may contain neurons that are
part of other identified structures like the parapyramidal neurons of
the raphe and the juxtafacial portion of nucleus paragigantocellularis
lateralis. Although the details of the anatomy of the RTN, including
its afferents, remain to be fully described, it is clear from
physiologically based experiments that the RTN contains
1) neurons that fire phasically with
respiratory output (22), 2)
chemoreception (5), 3) ionotropic and metabotropic glutamate receptors (16, 19),
4) cholinergic muscarinic receptors
(22), and 5) TRH receptors (the
present study). In anesthetized animals, lesions (20) or block of
glutamate (19) or muscarinic cholinergic (22) receptors decreases
phrenic activity and inhibits the response of the whole animal to
CO2. In awake animals, lesions
that affect ~35% of the RTN do not result in apnea but do decrease
the CO2 response by 39% (1). In
unanesthetized goats, surface medullary cooling at the RTN region
diminishes respiratory output at rest, with exercise, and in response
to hypercapnia and hypoxia (9). This small and recently identified region appears to have important influence on respiration and on
central chemosensitivity. Peptides also appear to play a role in the
function of the RTN. The location of substance P injections that
stimulate ventilation appears to include the RTN region (3). The
present study shows, with specific and focal RTN injection of TRH, a
strong stimulation of breathing and, in some cases, of blood pressure.
Natural source of TRH for the RTN.
Several medullary nuclei in the proximity of the RTN contain TRH-like
immunoreactivity, including the motor nucleus of VII, raphe magnus,
gigantocellular reticular cells, and parapyramidal neurons (2, 7, 14,
25). These would represent nearby sources of TRH input to the RTN
because local connections appear to be present at this region (30). NTS
and dorsal raphe (2) also contain TRH-like immunoreactivity and have
likely connections to the RTN region.
TRH receptors in the RTN.
There is no clear demonstration of TRH-receptor binding specific for
the RTN. TRH receptor studies of the medulla show their presence near
to, if not within, the RTN region, but the level of detail in the
provided pictures prevents a clear conclusion (17). In a beautiful
study, Sun et al. (27) showed the presence of synaptic boutons
containing TRH-like immunoreactivity in ventral medullary neurons
identified physiologically by their firing pattern to be respiratory
and characterized anatomically by intracellular biotin injection. These
neurons are not within the RTN per se, but their processes extend into
and through the RTN region. It is possible, then, that some of our RTN
TRH injection effects were on these ventral respiratory group neurons
via the TRH synapses on their extensive processes.
Mechanisms of TRH effects on neurons.
The response to TRH was sustained for prolonged periods, up to 4.5 h in
some cases. Earlier studies in our laboratory have also demonstrated a
sustained PNA response to metabotropic glutamate-receptor agonists that
lasted for 75-240 min (16) and to glutamate injected over a 60-s
period (16). Li and Nattie (16) hypothesized that the sustained effects
on respiration were the result of the activation of the metabotropic
glutamate receptor-associated signaling cascade, with subsequent
prolonged effects on neuronal activity. A similar hypothesis may be
applied in the present case. The TRH receptor is coupled to a G
protein, and the activation of this signaling pathway may result in
protracted changes in neuronal activity that lead to increased PNA.
In vitro, TRH alters the excitability of several neuronal populations,
including rat dorsal motor nucleus of the vagus, NTS, and hypoglossal
motoneurons (2, 8, 10, 23, 24). TRH increases the excitability of
neurons by inactivating K+
channels that are normally active at the resting membrane potential (2). The heightened excitability of the neurons was maintained for
5-40 min after TRH removal (2). Our results support the hypothesis
that TRH produces sustained changes in neuronal excitability, but these
in vitro studies do not explain the very long duration of our higher
TRH dose effects in vivo.
TRH effects on respiration at other sites.
Injections of TRH at more caudal medullary locations, including the
midline raphe and the NTS (18) and the pre-Bötzinger region in
the neonatal rat brain stem preparation (10), also result in strong
stimulation of respiratory output. Studies during development emphasize
the relative amounts of TRH mRNA, TRH-receptor binding, and neuronal
excitability in newborn rats with expression of adult values for these
variables occurring by 3 wk of neonatal life (2, 8). These observations
plus the effects on frequency of TRH injections into the neonatal rat
brain stem pre-Bötzinger region (10) raise the possibility that
TRH plays a role in rhythm generation, perhaps as a tonic excitatory
influence. Reckling et al. (24) described a direct postsynaptic action
of TRH depolarizing a subset of inspiratory neurons in the ventral
respiratory group region near the pre-Bötzinger region, data that
support this possibility.
Physiological significance.
The overall physiological significance of these results will not be
clear until we can identify a TRH-receptor antagonist or determine the
naturally occurring TRH concentrations. The presence of strong
TRH-induced effects on blood pressure and on breathing raises the
possibility that it plays a role in a generalized response pattern
involving coordination of a number of physiological responses. For
example, this could be viewed as a way to control or initiate the
"flight or fight" response.
ACKNOWLEDGEMENTS
The authors thank Holly Beeman for excellent contributions to preliminary stages of these experiments.
FOOTNOTES
Address for reprint requests: E. E. Nattie, Dept. of Physiology, 712E Borwell, Dartmouth Medical School, Lebanon, NH 03757-0001 (E-mail: Eugene.Nattie{at}Dartmouth.EDU).
Received 6 March 1997; accepted in final form 30 April 1997.
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