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Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Thyrotropin-releasing hormone (TRH) injected into the
retrotrapezoid nucleus (RTN) of anesthetized rats produces a large, prolonged stimulation of ventilatory output (C. L. Cream, A. Li, and E. E. Nattie. J. Appl. Physiol. 83: 792-799, 1997). Here we inject or dialyze TRH into the RTN of conscious rats. In 6 of 17 injections (200 nl, 3.1 ± 1.7 mM), ventilation
(
E)
increased 31% by 10 min, with recovery by 60 min. With dialysis, each
animal of one group (n = 5) received,
in random order, 10 mM TRH, 10 mM TRHOH (a metabolite of TRH), and
artificial cerebrospinal fluid (aCSF); each animal of a second group
(n = 5) received aCSF and 1 mM TRH.
TRHOH and aCSF had no sustained effects. TRH (1 mM) increased
E (32%,
P < 0.02, by 10 min, with recovery
by 60 min), O2 consumption
(O2; 19%,
P < 0.03), and body (rectal)
temperature (Tre; 0.5°C,
P < 0.09). TRH (10 mM) increased
E (78%,
P < 0.01, by 10 min, with no
recovery at 60 min), O2
(48%, P < 0.01), and
Tre (1.0°C,
P < 0.01). TRH also induced arousal.
The tissue volume affected in dialysis, estimated by spread of dialyzed
fluorescein (332.3 mol wt, mol wt of TRH = 362.4), was 1,580 ± 256 nl for 10 mM (n = 5) and 590 ± 128 nl for 1 mM (n = 5). We conclude that 1) the RTN is involved in the
integration of
E,
O2,
Tre, and arousal and
2) TRH may establish the
responsiveness of RTN neurons.
ventilation-metabolism coupling; fight-or-flight response; arousal; control of breathing
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE RETROTRAPEZOID NUCLEUS (RTN), one of several brain
stem nuclei that comprise the respiratory control network, lies in the
rostral ventrolateral medulla ventral to the facial nucleus (22).
Destruction of the RTN in anesthetized and decerebrate animals reduces
resting ventilation
(E) and the
ventilatory response to systemic
CO2 stimulation (14, 16), and
focal acidic stimulation of the RTN increases ventilatory output (4),
suggesting that the RTN provides two sources of input to the
respiratory control network: tonic and chemosensory.
Neurons of the RTN are responsive to several neurotransmitters, including glutamate and ACh (5, 13, 15, 17). Microinjections of glutamate or ACh agonists into the RTN increase phrenic activity; antagonists decrease it. Injection of glutamate over a prolonged period (60 s) or injection of a metabotropic glutamate receptor agonist results in increased phrenic activity, which is sustained for 50-60 min (17). Thus RTN neurons are capable of initiating large and sustained increases in phrenic activity after brief exposure to certain neuroactive substances, and glutamate and ACh appear to be tonically released in the RTN.
The neuropeptide thyrotropin-releasing hormone (TRH) has been
identified in various nuclei throughout the central nervous system
(CNS), including the caudal raphe (11, 26). TRH has excitatory effects
on many CNS functions, including arousal, body (rectal) temperature
(Tre), blood pressure,
metabolism, and
E (8, 18). TRH
administered through the cerebral ventricles increases
E in
anesthetized and conscious animals (8, 25). The RTN receives strong
input from the caudal raphe (unpublished observations), in which
25-30% of the neurons contain immunoreactive TRH (ir-TRH) (10,
24). Additionally, ir-TRH has been identified in the region of the RTN
(24). Therefore, we hypothesized that injection of TRH into the RTN
would increase
E.
This was affirmed in anesthetized, paralyzed, ventilated rats, inasmuch as unilateral microinjections of TRH into the RTN produced large and long-lasting increases in phrenic activity (6). TRH (0.25-10 mM, 10 nl) increased phrenic activity up to 150% above baseline, and the response duration often exceeded 4 h. At higher concentrations, mean arterial pressure was also increased in approximately one-half of the cases. We concluded that TRH acts in the RTN to increase ventilatory activity, an effect that may be part of a generalized autonomic reflex mechanism, such as the fight-or-flight response.
The present study was performed to determine the effects of TRH
administration into the RTN of the conscious rat. The effects of
disruption of the RTN on resting
E and
CO2 responsiveness in anesthesia
are different from the effects during wakefulness (1, 14, 16).
Furthermore, the conscious animal has intact chemoreflex regulation of
breathing, it represents a "closed system," and it allows a test
of the physiological potency of unilateral TRH administration into the
RTN. We hypothesized that TRH would increase
E in the
conscious rat and that the response would be similar in magnitude and
duration to the response in the anesthetized rat.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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General preparation. Animals weighing 300-400 g at the time of surgery were anesthetized with ketamine (100 mg/kg im) and xylazine (20 mg/kg ip). The crown of the skull was shaved and sterilized with alcohol. The rat was placed into a Kopf stereotaxic holder, and for the injection experiments in 12 rats a 0.46-mm-OD stainless steel microinjection cannula was placed into the region of the RTN. For the dialysis experiments in 10 rats a 0.38-mm-OD dialysis guide tube (no. 11, CMA, Acton, MA) was inserted into the brain stem. The coordinates for probe placement were 2.2 mm caudal and 1.8 mm lateral from lambda and 10.6-10.8 mm below the dorsal surface. The steel cannula or guide tube was secured to the skull with cranioplastic cement, and the wound was sutured. For the dialysis experiments the abdominal surface was shaved and sterilized, an incision was made through the linea alba, and a sterile telemetric temperature probe (model TA-F20, Data Sciences, St. Paul, MN) was placed in the abdominal cavity. The incision was closed, and the animal was allowed to recover for 3-4 days.
On each experimental day between 0900 and 1500 the rat would be weighed, then gently held while the dummy cannula filling the guide tube was removed and the injection cannula or the dialysis probe was inserted into the guide tube. Animals were placed into a Pappenheimer-modified plethysmograph box (19) and allowed 30-40 min to acclimate. Control measurements were taken in room air. For the injection experiments, animals received TRH (200 nl, 0.5-10 mM), andE was
monitored by using the whole body plethysmograph. Measurements were
taken over a 20- to 30-s period at 0, 5, 10, 20, 30, 40, and 50 min.
O2 and
Tre were not monitored during this
experiment. Tre was measured by
rectal probe at the beginning and end of the experiment. There were 17 injections in 12 rats. For the dialysis experiments, baseline
E,
O2, and
Tre were measured. The dialysis
pump was run for 30 min at a speed of 4.5 µl/min, and measurements
were taken over a 20- to 30-s period at 0, 5, 10, 20, 30, 40, 50, and
60 min. In the dialysis experiments, each animal in one group
(n = 5) was subjected, on separate
days, to three dialysis conditions selected randomly: artificial
cerebrospinal fluid (aCSF), 10 mM TRH in aCSF
(pGlu-His-Pro-NH2, TRH; catalog
no. P-2161, Sigma Chemical), or 10 mM TRH-free acid in aCSF
(pGlu-His-Pro-COOH, TRHOH; catalog no. P-3905, Sigma Chemical). In the
other dialysis group (n = 5), each
animal was dialyzed with aCSF and 1 mM TRH. TRH and control testing
were separated by a 24- to 72-h recovery period. Separate rats were
dialyzed for 30 min with 1 mM (n = 5)
or 10 mM (n = 5) fluorescein dye (332 mol wt; catalog no. F-1300, Molecular Probes) to estimate the volume of
distribution of TRH.
The plethysmograph chamber used in these experiments is similar to the
setup described by Pappenheimer (19). The analog output of the pressure
transducer was recorded on a strip-chart recorder (model 1200, Honeywell) and a pulse code modulation system (model 3000A, Vetter).
The animal chamber is designed to operate at atmospheric pressure; thus
the inflow and outflow of inspired gas must be balanced to prevent
hyper- or hypobaric conditions in the box. The inflow air was
humidified, and the flow rate was controlled by a flowmeter (model
7491T, Matheson). The outflow port was connected to the in-house vacuum
system, and a high-resistance "bleed" of the outflow line
provided ~100 ml/min of outflow gas to the
O2 analyzer (model SA-3, Applied
Electrochemistry). The flow rate through the plethysmograph was
maintained at 
1.4 l/min to prevent
CO2 rebreathing. The
plethysmograph chamber was calibrated with 0.3-ml injections. The
chamber temperature was measured before each period of ventilatory data
collection. Rat Tre was measured at the beginning and end of each experiment in the injection group and
during each period of ventilatory data collection by use of the analog
output from the telemetric temperature probe in the peritoneal cavity
in the dialysis groups.
At the conclusion of the experiments, animals were killed, and the
medulla was quickly removed and placed in 4% paraformaldehyde until
the time of sectioning. Brain stems were frozen, then sectioned at
50-µm thickness with a Reichert-Jung cryostat. Sections were counterstained with cresyl violet to identify anatomic landmarks and
the site of dialysis probe placement with the assistance of a rat brain
atlas (20). The guide tubes were removed postmortem but before the
brain stem was removed and sectioned. Removal of the guide tubes
required manipulation and produced tissue disruption. This facilitated
the anatomic verification of guide tube and probe tip location but also
increased the volume of tissue disruption compared with that produced
by simple insertion. In the animals dialyzed with fluorescein, the
tissue was frozen and sectioned without fixative, then analyzed for fluorescence.
Respiratory data were transferred from the Vetter system to the DataPac
III software system. With use of the DataPac III system, a
breath-by-breath analysis was performed, with the pressure deflections and the respiratory cycle time for each breath being determined over a
20- to 30-s time period. The data were exported to Sigmaplot 4.0 (Jandel Scientific), and tidal volume
(VT) per 100 g body weight,
frequency, and E were
calculated for each breath.
O2 was determined by
subtracting the outflow O2
(constant flow rate 1.4-1.5 l/min) from the inflow
O2, dividing by body weight, and multiplying by 60 to obtain an hourly rate
(ml · g
1 · h1).
The inflow O2 content was
calibrated at the beginning of each experiment, and the flow rate of
gas was set. The outflow content of
O2 was read from the
O2 sensor.
The results for
E,
VT, frequency,
O2, and
Tre were compared within and
between each of the experimental groups with treatment (aCSF, 10 mM
TRH, 10 mM TRHOH and aCSF, 1 mM TRH) and time as factors with a one-way
and two-way repeated-measures ANOVA (Sigmastat, Jandel Scientific;
SYSTAT). A post hoc Tukey test was performed when significant
differences were found.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Controls.
Injections of aCSF had no significant sustained effect on
E, and only 6 of 17 injections of TRH resulted in an increase in
E of >10%
of baseline, an arbitrary definition of a response in these
injection experiments. With dialysis the responses of E,
O2, and
Tre to aCSF were small,
unsustained, and variable and did not differ between the two groups of
rats (n = 5 in each) that received
dialysis. These data have been pooled
(n = 10) and are shown as
the responses to aCSF dialysis. The responses of E,
O2, and
Tre to dialysis with the TRH
metabolite TRHOH (10 mM) were not significantly different from those
obtained with dialysis with aCSF. These data are not shown.
E.
Figure 1 shows the responses of
E, expressed
in absolute terms (A) and as percent
baseline (B), to the six injections
of TRH that produced a response. In these responders,
E increased to
31% of baseline at 10 min from a baseline value of 74 to 97 ml · min1 · 100 g1. By 60 min,
E returned to
baseline levels. The response included an increase in
VT and frequency. The average
TRH concentration of these six injections was 3.1 ± 1.7 mM.
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E, expressed
in absolute terms (A) and as percent
baseline (B), to dialysis in the RTN
of aCSF, 1 mM TRH, and 10 mM TRH. At 1 mM, TRH increased mean
E by 29 and
32% at 5 and 10 min from a control value of 68.5 to 88.3 and 90.3 ml · min1 · 100 g1, respectively. By 40 min, 10 min after dialysis was stopped, E returned to
within control values. This response was significant (P < 0.02) and included increases in
VT and frequency, although the
latter did not reach significance (P < 0.08). At 10 mM, TRH dialysis increased mean
E by 78% from
79.3 to 141.4 ml · min1 · 100 g1 after 5 min of dialysis.
The increase in
E was
significant (P < 0.01).
E remained
significantly elevated relative to aCSF for the remaining 55 min. The
early E
increase was mediated by an increase in frequency and
VT. The remainder of the
E response was
mediated by a continued increase in frequency and a decrease in
VT.
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O2.
Figure 3 shows responses of
O2, expressed in absolute
terms, to dialysis in the RTN of aCSF, 1 mM TRH, and 10 mM TRH. At 1 mM, TRH increased mean
O2 by 15 and 19%
at 5 and 30 min from a control value of 0.89 to 1.02 and 1.06 ml · h1 · g1,
respectively. This response was significant
(P < 0.03). By 50 min, 20 min after
dialysis was stopped, O2
returned to within control values. At 10 mM, TRH dialysis increased
mean O2 by 38% from 0.88 to
1.21 ml · h1 · g1
after 10 min of dialysis. O2
remained significantly elevated relative to aCSF for the remaining 55 min. The response was significant (P < 0.01).
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Tre. Figure 3 also shows the responses of Tre, expressed in absolute terms, to dialysis in the RTN of aCSF, 1 mM TRH, and 10 mM TRH. At 1 mM, TRH increased mean Tre by 0.5°C at 40 min from a control value of 38.1 to 38.6°C. This response was not significant (P < 0.09). At 10 mM, TRH dialysis increased mean Tre by 1.0°C from 37.7 to 38.7°C after 30 min of dialysis. Tre remained elevated relative to aCSF for the remaining 55 min. The response was significant (P < 0.01).
E and
O2.
Figure 4 shows
E and
O2 expressed as percent
baseline for 10 mM (A) and 1 mM
(B) TRH dialysis. Although there is
a suggestion that the increase in
E is greater
than the increase in O2 in
the first 5-10 min with 10 mM TRH dialysis, for the most part the
increase in E
seems to match the increase in
O2. There is
sufficient variability in the E and
O2 results that arterial blood-gas sampling is needed to show conclusively whether there is
hyperventilation.
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Arousal. We also made subjective evaluations of the arousal state of each rat. Normally, after the period of acclimatization in the plethysmograph, rats will rest quietly and no longer explore the chamber. After a time they will often appear to fall asleep, curling up with their eyes closed and lying motionless on the floor. We often needed to arouse them by gently tapping on the side of the chamber during control or TRHOH dialysis. We noted that with TRH treatments the rats became more aroused. They would not sleep, they would rise from the prone position, and they would stand on all four legs looking alert or once again exploring the chamber as during their initial acclimatization. They appeared to be more vigilant.
Anatomy.
Figure 5 shows single medullary cross
sections from each of the five rats in the group that was dialyzed with
1 mM TRH. Each cross section is approximately at the center of the site
at which the dialysis probe tip was located in vivo. The cresyl violet stain allows one to easily detect the tissue disruption produced by the
probe (rectangles). The size of the disruption is not indicative of the
region of tissue disruption in vivo, in that it was difficult to remove
the guide tube at the time the animals were killed without enlarging
the region of tissue damage. Nevertheless, this anatomic analysis shows
the location of the probe tip. In each case it resulted in TRH
application to the RTN during dialysis. The probe tips in the five rats
that were dialyzed with 10 mM TRH are similarly located (data not
shown), and the six injections of TRH shown in Fig. 1 are also
similarly located in the RTN (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Conscious rats,
E, and TRH.
The major findings in this study are that focal application of TRH into
the RTN region of the conscious rat increases
E, O2, and
Tre, as well as the level of
arousal. We used two methods to apply the TRH focally: microinjection
and microdialysis. Of 17 microinjections (200 nl), all in the RTN
region as judged by postmortem identification of fluorescent beads
mixed into the aCSF, only 6 increased
E. The mean
TRH concentration of these six injections was 3.1 mM. The ventilatory
response magnitude and time course for these six injections are similar
to those with dialysis over 30 min of 1 mM TRH (Figs. 1 and 2), with
recovery within 60 min in each case. However, with dialysis, all five
rats showed a response suggesting a larger region of TRH distribution. With dialysis of 10 mM TRH, again all five rats showed a response that
was greater and longer lasting than that after dialysis with 1 mM TRH
or the microinjections.
E by 235%
(vs. 78% in our study) and
O2 by 11.7-fold (vs. 48% in
our study). These quantitative differences are likely due to the route
of administration. Injection into the cerebral ventricles affects a
much larger region than our local dialysis.
In an earlier study in anesthetized rats with microinjection of TRH (20 nl, 0.5-10 mM) into the RTN, we observed substantial effects (up
to 110% increase in ventilatory output) that were long lasting (up to
4 h). The magnitude of this response is similar to that in the
conscious rats with 10 mM dialysis and much larger than that observed
with 1 mM dialysis or with the larger microinjections (200 vs. 20 nl)
of similar TRH concentrations in conscious rats. The responses in the
anesthetized rats also lasted longer, up to 4 h, than any of those in
conscious rats. This greater effectiveness of the TRH injections in
anesthetized rats is unexpected and difficult to explain. We expect
that our TRH delivery was at higher concentrations to a wider area in
the dialysis experiments, and in the conscious rat microinjection
experiments the injection volume was 10 times greater. Slower
metabolism and/or clearance of TRH under anesthesia or the presence of
suppressive influences from higher brain regions in the conscious rats
could be playing a role in these differences in response. Also the
anesthetized animals were ventilated to an end-tidal
CO2 that was just above the apneic
threshold. Consequently, the amplitude of the baseline phrenic burst
was relatively small and may have had a larger margin for response than
did VT in the conscious animal.
Finally, there is evidence that suggests a greater physiological role
of the RTN region in anesthesia. Lesions of the RTN in the anesthetized
rat decrease eupneic respiration, often to apnea, and
CO2 chemosensitivity, often
abolishing it (16), whereas lesions in conscious rats do not affect
eupnea and decrease CO2
responsiveness only 39% (1). It seems plausible that RTN function is
arousal state dependent, and arousal state modulates the level of
response to any given RTN manipulation.
Our study in the anesthetized rat (6) also suggested that
subpopulations of neurons within the RTN are responsive to TRH. When an
injection was ineffective, we could easily reposition the pipette to
find a reactive site because of the ventral approach used in those
experiments. That only 6 of 17 injections in the conscious rats
produced a response supports this interpretation. In fact, we switched
to the microdialysis technique to establish a concentration gradient
that would facilitate exposure of more neurons of the RTN to TRH in
each trial without the tissue disruption produced by large injections.
We appear to have achieved this, inasmuch as all animals treated by
dialysis responded to TRH.
O2 and
Tre.
O2 increased significantly in
response to dialysis with 1 or 10 mM TRH; it was not measured in the
microinjection experiments. The mechanism of the increased metabolic
rate is unclear, although such an effect was also observed in prior
studies with intracerebral TRH administration (26). In our case, the
TRH application was focal, and we still observed these significant
changes in metabolic rate.
O2. The time
courses of the Tre and
O2 responses were similar.
Unpublished findings from our laboratory support a possible direct
thermoregulatory action of TRH in the RTN. Retrograde tracing studies
demonstrate that the RTN receives input from the raphe magnus and the
subceruleus. Both of these nuclei receive and integrate thermoreceptor
input and, when stimulated, are capable of producing hyperthermia (3, 9). The RTN may aid in the integration of this information and couple
the thermogenic response to the appropriate level of E.
In homeotherms,
E,
Tre, and
O2 are tightly linked. For
example, exposure to cold temperatures activates thermogenic responses; this increases O2, and
E increases to
meet the increased demand for O2
(12). The sites of this integration have been undefined; we suggest
that the RTN may be one of these sites.
TRH and arousal. TRH can antagonize the effects of various anesthetics (18, 23). TRH administered intravenously or into the cerebral ventricles decreases anesthetic-induced narcosis and hypothermia (23). A possible site of this TRH action is the brain stem reticular formation (27). Intravenous TRH increases the firing rate of reticular field neurons and decreases the threshold for arousal changes in the frontal cortex (26).
In the conscious rats we noted that during aCSF or TRHOH dialysis the animals would quickly acclimate to being in the plethysmograph and enter sleep, as defined by behavioral criteria. When TRH was dialyzed in these same animals, they did not sleep. They would remain upright on all fours and, with the 10 mM dose, would appear hypervigilant. This effect of TRH on arousal was unexpected, and it may have contributed to the increase inO2. If arousal and O2 are increased,
Tre and
E may have
increased as a necessary result.
Tissue spread with dialysis. With microdialysis, it is difficult to determine the tissue concentration profile of TRH. It appears certain that the concentration in the dialysate is much higher than that detected outside the probe. Alessandri et al. (2) found 35% delivery into the tissue of glutamate (10-1,000 mM at 2.5 µl/min) during the first 30 min of dialysis. If applied to our case, the TRH concentration next to the probe would be 0.35-3.5 mM for our 1 and 10 mM doses. We used a higher flow rate, 4.5 µl/min, so this estimate is likely to be on the high side. In a study using two dialysis probes, one for delivery and the other for collection and measurement, the steady-state concentrations of drugs 1.5 mm away from the probe were 10-100 times lower than the concentration in the probe (7). Neither of the drugs studied are metabolized in the CNS. TRH, on the other hand, is metabolized, and its concentration drop would be expected to be greater and its spread less. Even so, using these data, for the 1 mM dose we could expect maximum TRH concentrations of 0.35 mM at the probe and between 0.01 and 0.1 mM at 1.5 mm from the probe. For the 10 mM dose, these would be 3.5 mM at the probe and between 0.1 and 1.0 mM at 1.5 mm from the probe.
Our experimentally measured estimate of the tissue volume affected by TRH comes from dialysis of a fluorescent molecule with approximately the same molecular weight as TRH (332.3 vs. 362.4). Because this molecule is not metabolized, the volume of distribution for TRH is arguably smaller. The average (n = 5) volume of dye distribution when dialyzed at 1 mM was 590 nl, the average rostral-to-caudal length of the fluorescein distribution was 870 µm, and the average radius of fluorescein distribution at the cross section with the largest area of distribution was 555 µm. Thus, for 1 mM TRH dialysis, using the fluorescein distribution, we estimate a spread of 555 µm radially in the plane of the cross section and 435 µm in the rostral or caudal direction. For 10 mM TRH dialysis (n = 5) the fluorescein distribution volume was 1,580 nl, with a rostral-to-caudal length of 1,400 µm and an average radius of 704 µm at the cross section with the largest area. For the 10 mM TRH dialysis, we estimate a spread of 704 µm radially in the plane of the cross section and 700 µm in the rostral or caudal direction. The volume of distribution for the larger dose, 10 vs. 1 mM, was almost three times greater; the radial spread was 27% greater. Our estimate of spread within the medulla, as measured by dialysis and detection of fluorescein dye, is less than that deduced from published tissue concentrations of other substances dialyzed in other brain regions (7). With use of either approach, the distribution of TRH with dialysis of 1 mM TRH is likely to be largely, if not entirely, within the RTN region. With the 10 mM dose, the TRH is focused in the RTN region, but its spread probably involves contiguous regions, including the facial nucleus, the juxtafacial portion of the nucleus paragigantocellularis lateralis, the parapyramidal region, and possibly the medullary raphe. We believe that the microdialysis approach is a useful method for administration of neuroactive substances to specific brain sites in a conscious animal model. The probe tip containing the semipermeable membrane is 1 mm long and 240 µm diameter, with a volume of 45 nl. Thus its size and the region of tissue disruption are similar to that after an injection of this volume. On the basis of the reliability of the physiological responses and the distribution of the fluorescein dye after dialysis, the volume of tissue affected is greater than that affected by a 10- to 20-nl injection or even a 200-nl injection. Once the probe tip is in place, no further tissue disruption takes place, and repeated dialyses can be performed in the same animal. The major problem with this technique is the delineation of the exact region in the tissue that is affected by the dialyzed substance and the determination of the concentration of the substance at various distances from the probe. This problem also exists for the microinjection approach.Physiological significance. The RTN receives input from respiratory, raphe, and reticular sources (unpublished observations) and has efferent connections with the respiratory control network and the limbic system (22, 28). Although the RTN has not been identified as a major site containing ir-TRH or TRH receptors, several sources of RTN input contain ir-TRH and are probable TRH connections to the RTN. Approximately 25-30% of serotonin neurons of the caudal raphe contain ir-TRH and represent the most likely local source of TRH to the RTN (11, 26).
At the cellular level, TRH inactivates K+ channels normally active at the resting membrane potential, which depolarizes the neurons and increases membrane responsiveness (21). This action amplifies the neural response to afferent inputs and may increase subsequent motor output. The physiological settings in which TRH is normally released are unknown. The activity of the caudal raphe is arousal state dependent, being highest with wakefulness and lowest or absent during rapid-eye-movement sleep (10). The change in activity level is presumably associated with variations in the amount of the neurotransmitters released, including TRH. Decreased TRH in the RTN would lead to relative hyperpolarization of the neurons and diminished neuronal activity. This may be manifest most clearly during rapid-eye-movement sleep whenE,
Tre, and O2 become uncoupled, during
presumed RTN relative inactivity.
The behavioral arousal effects of TRH in the RTN cannot be adequately
explained from our present experiments. Anesthetized rats treated with
TRH in the RTN required more frequent additions of anesthesia than in
our standard laboratory protocol, and conscious rats did not sleep when
dialyzed with TRH in the RTN. This may reflect direct actions of RTN
neurons on the reticular activating system to increase arousal.
Alternatively, the arousal effects may be secondary to
O2,
Tre, and
E changes
initiated in the RTN and coupled to arousal at a remote site.
We conclude that TRH in the RTN can alter the integration of
E,
Tre,
O2, and arousal. The lack of
a TRH receptor antagonist precludes us from blocking any effects of
endogenous TRH, but these striking responses in conscious rats hint at
an important role for TRH, possibly as a state-dependent modulator of
the control of breathing.
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ACKNOWLEDGEMENTS |
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This research was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-28066. C. Cream was supported by NHLBI Grant HL-28066, Minority Supplement, and Respiratory Training Grant HL-07449. Gerard Gagne and Ross Downey, who were supported by NHLBI Medical Student Summer Research Training Grant HL-07715, helped with the development of the flow-through plethysmograph system.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: E. Nattie, Dept. of Physiology, Borwell Bldg., Dartmouth Medical School, Lebanon, NH 03756-0001 (E-mail: Eugene.Nattie{at}Dartmouth.edu).
Received 30 July 1998; accepted in final form 15 April 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Akilesh, M. R.,
M. Kamper,
A. Li,
and
E. E. Nattie.
Effects of unilateral lesions of retrotrapezoid nucleus on breathing in awake rats.
J. Appl. Physiol.
82:
469-479,
1997
2.
Alessandri, B.,
H. Landolt,
H. Langemann,
J. Gregorin,
J. Hall,
and
O. Gratzl.
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