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Am J Physiol Regul Integr Comp Physiol 290: R1654-R1663, 2006. First published January 12, 2006; doi:10.1152/ajpregu.00704.2005
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Orexin neuron-mediated skeletal muscle vasodilation and shift of baroreflex during defense response in mice

Wei Zhang,1 Takeshi Sakurai,2,3 Yasuichiro Fukuda,4 and Tomoyuki Kuwaki1,4

Departments of 1Molecular and Integrative Physiology and 4Autonomic Physiology, Chiba University Graduate School of Medicine, Chiba-City, Chiba; 2Department of Molecular Pharmacology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba; and 3Exploratory Research for Advanced Technology Yanagisawa Orphan Project, Japan Science and Technology Corporation, Tokyo, Japan

Submitted 3 October 2005 ; accepted in final form 9 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that some features of the defense response, such as increases in arterial blood pressure (AP), heart rate (HR), and ventilation were attenuated in prepro-orexin knockout (ORX-KO) mice. Here, we examined whether the same was true in orexin neuron-ablated [orexin/ataxin-3 transgenic mice (ORX/ATX-Tg)] mice. In addition, we examined other features of the defense response: skeletal muscular vasodilation and shift of baroreceptor reflex. In both anesthetized and conscious conditions, basal AP in ORX/ATX-Tg mice was significantly lower by ~20 mmHg than in wild-type (WT) controls, as was the case in ORX-KO mice. The difference in AP disappeared after treatment with an {alpha}-blocker but not with a beta-blocker, indicating lower sympathetic vasoconstrictor outflow. Stimulation of the perifornical area (PFA) in urethane-anesthetized ORX/ATX-Tg mice elicited smaller and shorter-lasting increases in AP, HR, and ventilation, and skeletal muscle vasodilation than in WT controls. In addition, air jet stress-induced elevations of AP and HR were attenuated in conscious ORX/ATX-Tg mice. After pretreatment with a beta-blocker, atenolol, stimulation of PFA suppressed phenylephrine (50 µg/kg iv)-induced bradycardia ({Delta}HR = –360 ± 29 beats/min without PFA stimulation vs. –166 ± 26 during stimulation) in WT. This demonstrated the resetting of the baroreflex. In ORX/ATX-Tg mice, however, no significant suppression was observed (–355 ± 16 without stimulation vs. –300 ± 30 during stimulation). The present study provided further support for our hypothesis that orexin-containing neurons in PFA play a role as a master switch to activate multiple efferent pathways of the defense response and also operate as a regulator of basal AP.

hypothalamus; stress; blood pressure; respiration

OREXIN A AND B, also known as hypocretin 1 and 2, respectively, are hypothalamic neuropeptides. They are cleaved from a common precursor molecule, prepro-orexin, undergoing proteolytic processing (33, 41). Although the localization of orexin-containing cell bodies is restricted to the hypothalamus, the orexin-containing fibers and terminals are widely distributed in the hypothalamus, thalamus, cerebral cortex, circumventricular organs, brain stem, and spinal cord (13, 28). This anatomic feature establishes the basis that orexin contributes to multiple physiological functions, including feeding behavior, energy homeostasis, sleep-wake cycle, and regulation of the autonomic and neuroendocrine systems (25, 36, 41).

At present, there are two genetically engineered mice models of orexin deficiency to study possible roles of intrinsic orexin in physiological functions mentioned above. One is the prepro-orexin knockout (ORX-KO) mouse that was developed by a conventional knockout technique (4), and another is the orexin neuron-ablated mouse (17). The latter was developed using a transgenic technique by introducing a truncated Machado-Joseph disease gene product, ataxin-3, with an expanded polyglutamine stretch under the control of the human orexin promoter. In these orexin/ataxin-3 transgenic mice (ORX/ATX-Tg), orexinergic neurons are selectively and postnataly degenerated and suffer >99% loss at 4 mo of age. In these mice, not only orexin, but also other neuropeptides or modulatory factors contained in the orexinergic neurons, such as dynorphin, galanin, and glutamate, are considered deficient (6). Although both ORX-KO and ORX/ATX-Tg mice showed a phenotype strikingly similar to human narcolepsy (4, 17), autonomic phenotype of the latter has not been examined.

In our previous studies, we have demonstrated by using ORX-KO mice that endogenous orexin contributes to eliciting some features of the defense response, such as increases in arterial blood pressure (AP), heart rate (HR), ventilation, cortical arousal, physical activity, and stress-induced analgesia (22, 40). In addition, basal AP in ORX-KO mice was lower by ~20 mmHg than that in wild-type (WT) controls probably through lower sympathetic vasomotor tone because an {alpha}-blocker, prazosin, cancelled the difference in AP. Exogenous application of orexin increased AP and ventilation in WT mice (44). However, two other important features of the defense response have not yet been analyzed. Those are skeletal muscle vasodilation and attenuation of reflex bradycardia or resetting of the baroreflex (11, 23, 29, 35, 38).

The aim of this study was to examine whether orexin also contributes to redistribution of blood flow and suppression of reflex bradycardia during the defense response using ORX/ATX-Tg mice. We used ORX/ATX-Tg mice in the present experiment so that we might gain some insight into the physiological significance of the other neuropeptides or modulatory factors contained in the orexinergic neuron by comparing present data with our previous data using ORX-KO mice (22).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

ORX/ATX-Tg mice with mixed genetic background of C57BL/6-DBA2 (17) were over five times backcrossed to C57BL/6. They were maintained in heterozygotes and crossed with C57BL/6 to obtain ORX/ATX-Tg mice and WT littermates. Experiments were performed in a manner blinded to genotypes. The genotype of ORX/ATX-Tg mice was identified by PCR of DNA extracted from the tail as had been reported (17). Animals used in this study were male ORX/ATX-Tg mice (n = 22) and WT (n = 21) mice. Mice had food (standard chow; MF, Oriental Yeast, Tokyo, Japan) and tap water available ad libitum. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Animal Use Committee of Chiba University Graduate School of Medicine.

Experiment 1: Chemical Stimulation of the PFA

General preparation of anesthetized mice. Mice were anesthetized with intraperitoneal injections of urethane (1.3 g/kg). Adequacy of anesthesia was judged by stability of baseline AP, HR, respiration, and blood flow throughout the experiment. The animal was placed in the prone position in a stereotaxic frame (model ST-7; Narishige, Tokyo, Japan) so that bregma and lambda would be horizontal. A small hole was drilled in the skull for insertion of a metal electrode or a glass micropipette into the hypothalamus. After completion of surgery, at least 1 h was allowed to pass to stabilize all parameters. Throughout the experiment, rectal temperature was kept constant at 37.0 ± 1.0°C by a heating pad connected to a thermo controller (model ATB-1100; Nihon Kohden).

Measurement of cardiorespiratory parameters in anesthetized mice. AP, HR, and ventilation were recorded as described in our previous report (22, 44). In brief, AP was measured by a polyethylene tubing placed in the abdominal aorta through an incision of the right femoral artery. Mean AP (MAP) was calculated by damping (0.2 Hz low pass) the phasic AP signal. HR was recorded using a HR counter triggered by the AP pulse. Respiratory flow signal was obtained through a Lilley-type pneumotachograph connected to the tracheal tubing.

Two laser Doppler flow probes (model BRL-100; Bio Research Center, Tokyo, Japan) were implanted into the mesentery of the ileum and the left gastrocnemial muscle. The visceral probe was inserted from a small incision made in the abdominal wall. The precise position of the probe tip was confirmed after each experiment. The muscular probe was inserted to a depth of ~1 mm from the fascia. Flow signals were low-pass filtered at 0.1 Hz to avoid possible movement artifacts (26). Because stimulation of the perifornical area (PFA) induced changes in both blood flow and pressure, vascular conductance was calculated as blood flow divided by MAP.

Chemical stimulation of the PFA to elicit defense response. The same procedure was used in our previous study (22). In brief, a glass micropipette was stereotaxically inserted into the PFA where orexin neurons are located (13, 28), and the defense response was most effectively elicited (22): 2.0 mm caudal to the bregma, 0.65 mm lateral to the midline, 5.0 mm ventral to the bregma. The micropipette was filled with a GABA-A receptor antagonist, bicuculline methiodide (0, 0.1, 0.3, 1.0 mM), dissolved in artificial cerebrospinal fluid (ACSF). While fluid meniscus in the micropipette were observed, a volume of 20 nl was injected. On each animal, bicuculline was administered sequentially from the lowest dose to the highest after recorded parameters returned to baseline. Position of the pipette tip was verified histologically as had been described (22).

Experiment 2: Baroreflex During PFA Stimulation

Assessment of baroreceptor reflex in anesthetized mice. AP and HR were measured as described in Measurement of cardiorespiratory parameters in anesthetized mice. An additional polyethylene tubing (model SP8; Natume, Tokyo, Japan) was inserted into the left femoral vein for drug delivery. Respiration and blood flow were not measured in this experiment. To observe vagally mediated reflex bradycardia, the animals were pretreated with intraperitoneal injection of a beta blocker, atenolol (5 mg/kg). In our preliminary experiment, this dose was shown to be sufficient to suppress tachycardia induced by the beta-stimulant, dobutamine (10 µg/kg iv), for more than 2 h after injection. After administration of atenolol, 20–30 min were allowed to stabilize all parameters. Baroreflex bradycardia was induced by a rise in AP with an intravenous injection of phenylephrine (0.1 mg/ml x 0.5 µl/g). The PFA was activated by either electrical or chemical stimulus. For electrical stimulation, the tip of a stainless steel electrode was positioned in the PFA. Stimulation was a train of rectangular pulses of 0.3 mA, 0.5 ms duration at 100 Hz for 30 s. Chemical stimulation was applied as mentioned above using 1.0 mM of bicuculline. In each animal, phenylephrine was injected three times; >20 min after administration of atenolol without any stimulus to PFA, during electrical stimulation to PFA, and ~5 min after injection of bicuculline when the rise in AP was at maximum.

Experiment 3: Conscious Animals

Measurement of cardiovascular parameters and application of air jet stress to elicit defense response in conscious mice. After the mice were anesthetized with isoflurane (2–3%), a polyethylene tubing was inserted into the femoral artery to measure AP, and wire electrodes were attached to the root of the legs to measure cardiovascular parameters with an ECG. The animal was loosely restrained in a plastic restrainer (height x width x length = 3.0 x 3.0 x 7.0 cm) in which the animal could rotate around the rostrocaudal axis but could not turn rostrocaudally. Experiments began after a 3-h period of recovery from anesthesia. First, AP and ECG were continuously monitored for 20 min, and these data were used for the assessment of the baroreflex (see Assessment of baroreceptor reflex in conscious mice by a naturally occurring sequence method). An air jet was then applied, which consisted of pulses (2-s duration delivered every 10 s for 5 min) of compressed air aimed at the forehead from a nozzle with a 1-mm opening that was positioned 1 cm away from the animal’s nose.

Pharmacological interventions. Thirty minutes after the completion of the above experiment, mice were sequentially treated with intraperitoneal injections of atenolol (5 mg/kg; a beta blocker) and prazosin hydrochloride (1 mg/kg; an {alpha}1-adrenergic receptor antagonist) to assess cardiac and vasoconstrictive sympathetic nerve activity. The peak AP and HR values within 20 min after the administration were recorded.

Assessment of baroreceptor reflex in conscious mice by a naturally occurring sequence method. The procedure described in our previous study was used (21). In brief, recordings of AP were scanned by a computer program developed at our institution to identify the spontaneous sequences of three or more consecutive beats in which systolic AP (SAP) progressively increased or decreased by more than 1 mmHg per beat. Of these SAP sequences, those that were associated with baroreflex-driven lengthening or shortening in RR intervals (more than 0.5 ms, twice the analog-to-digital conversion interval) were selected and defined as baroreflex sequences. A linear regression analysis between SAP and RR interval was applied to each baroreflex sequence, and the slope of the regression line was calculated only when the coefficient of determination exceeded 0.8. An average value of the slopes in a mouse was taken as the gain of baroreflex in the animal. We also calculated the baroreflex effectiveness index, defined as the ratio between the number of baroreflex sequences and the total number of SAP ramps, regardless of the possible occurrence of concomitant reflex change in RR intervals (10).

Data Analysis and Statistical Procedure

All signals were fed into a personal computer after analog-to-digital conversion (MacLab; AD Instruments, Bella Vista NSW, Australia) together with event signals.

In the first series of the experiments (chemical stimulation to PFA), data were analyzed as described in our previous paper (22). In brief, data were averaged by the minute, and the baseline value was defined as the mean value during the 5 min before the injection of bicuculline. As a respiratory parameter, respiratory minute volume (frequency x tidal volume/body wt) was calculated using a signal-processing software, Chart (AD Instruments). Areas under the curve (AUC) above baseline values were calculated during periods of 10, 15, 20, and 30 min from the injection for 0, 0.1, 0.3, and 1.0 mM of bicuculline, respectively. The recovery time was defined as the time from injection to the time for MAP, HR, or respiratory minute volume to return to a value 5% above the baseline.

Responses in AP and HR to phenylephrine were expressed as the mean during 2 s around the peak effect. In the last series of the experiments that used conscious mice and air jet stress, AP and HR data were averaged by the minute. AUC of the 5-min stimulation period was calculated.

All data were expressed as means ± SE. Effects of microinjection of bicuculline, intravenous phenylephrine, or intraperitoneal atenolol and prazosin were assessed by repeated-measures ANOVA. Post hoc comparisons of the Student-Newman-Keuls procedure or Student’s unpaired t-test were used to compare between genotypes. A contrast test was used to compare between treatments within the genotype. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Animals

Table 1 summarizes body weights and ages of the animals used in this study. Body weights were clearly different between ages but not significantly different between the genotypes under C57BL6 background, although those of the mutants tended to be higher than the WT littermates. Difference in generation of backcrossing to C57BL/6 seemed not to have affected the body weight.


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  		Table 1. Experimental animals used in this study

 
Experiment 1: Effect of PFA Chemical Stimulation in ORX/ATX-Tg mice

The defense response is characterized not only by increases in AP, HR, and ventilation but also by shift of blood flow from visceral to skeletal vasculature. To test our hypothesis that orexin may contribute to expression of all the features of the defense response, we compared the effects of chemical stimulation of PFA by bicuculline between ORX/ATX-Tg and WT mice. Before stimulation, the average values of MAP, HR, and respiratory minute volume in eight WT mice were 105 ± 4 mmHg, 593 ± 15 beats/min, and 0.99 ± 0.11 ml·min–1·g–1, respectively. In nine ORX/ATX-Tg mice, basal values of MAP, HR, and respiratory minute volume were 88 ± 2 mmHg, 625 ± 11 beats/min, and 1.03 ± 0.09 ml·min–1·g–1, respectively. The MAP was significantly (P < 0.05, Student’s t-test) lower than that in WT mice (Fig. 1). Basal HR and respiratory minute volume were not significantly different between the genotypes.


Figure 1
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  		Fig. 1. Effects of microinjection of bicuculline methiodide to the perifornical area (PFA) in wild-type (WT) mice and orexin/ataxin-3 transgenic mice (Tg) on arterial blood pressure (AP), heart rate (HR; in beats/min), respiratory minute ventilation, and the muscular and the visceral vascular conductance. Arrows indicate timing of microinjection of bicuculline (20 nl). Data are presented as means ± SE of 8 WT mice and 9 Tg mice.

 
In WT animals, microinjection of 0.1–1 mM of bicuculline to the PFA elicited dose-dependent increases in MAP, HR, and respiratory minute volume (Fig. 1), while ACSF (vehicle) did not cause any significant changes, as was the case in our previous report (22). An injection of bicuculline at 0.1, 0.3, and 1.0 mM induced peak increases in MAP (17 ± 4, 28 ± 5, and 47 ± 7%, respectively), HR (5 ± 2, 6 ± 1, and 8 ± 2%, respectively), and respiratory minute volume (77 ± 17, 99 ± 21, and 179 ± 26%, respectively), which were all statistically significant (repeated-measures ANOVA followed by the contrast test). Although bicuculline increased both muscular and visceral blood flow, simultaneous increases in MAP were smaller than the changes in muscular blood flow and greater or similar to those in visceral blood flow. Consequently, vascular conductance in the muscle increased dose dependently, whereas visceral conductance did not change or even decreased (Fig. 1). Peak increases in vascular conductance in the muscle by 0.1, 0.3, and 1.0 mM of bicuculline (55 ± 18, 57 ± 22, and 86 ± 21%, respectively) were all statistically significant, whereas peak changes in vascular conductance in the viscera (1 ± 9, 14 ± 9, and –15 ± 6%, respectively) did not reach statistical significance (repeated-measures ANOVA followed by the contrast test).

There was a significant quantitative difference between ORX/ATX-Tg mice and WT in responses to bicuculline. Whereas 0.1 mM of bicuculline was effective to elicit significant changes in MAP, HR, respiratory minute volume, and vascular conductance in the muscle in WT, the same dose of bicuculline did not cause any significant changes in ORX/ATX-Tg mice (Fig. 1, left) except for a small and short-lasting (Table 2) increase in MAP. At the dose of 0.3 mM, cardiorespiratory responses in ORX/ATX-Tg mice were smaller and shorter lasting than those in WT (Fig. 1, middle, Table 2). At the dose of 1.0 mM, responses in AP, respiratory minute volume, and vascular conductance in the muscle were still smaller and shorter lasting, although responses in HR were comparable between the two genotypes (Fig. 1, right, Table 2). Consequently, response magnitudes, as calculated by the AUC, were significantly smaller (repeated-measures ANOVA followed by the post hoc comparisons of Student-Newman-Keuls procedure) in ORX/ATX-Tg mice for all the parameters (Fig. 2) except for vascular conductance in the viscera in which no significant change was observed even in WT.


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  		Table 2. Duration of responses by bicuculline

 

Figure 2
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  		Fig. 2. Effects of microinjection of bicuculline methiodide to the PFA in WT and Tg mice on AP, HR, respiratory minute ventilation, and the muscular and the visceral vascular conductance. Changes in these parameters are expressed as area under the curve (AUC; see also MATERIALS AND METHODS). Data are presented as means ± SE of 8 WT mice and 9 Tg mice. *P < 0.05; **P < 0.01 vs. WT mice.

 
Histological examination revealed that there was no difference in the dye distribution between WT and ORX/ATX-Tg mice, and all of the injections were successfully made in the dorsal part of the PFA (not shown).

Experiment 2: Baroreceptor Reflex During PFA Stimulation

Another feature of the defense response examined in this study was the resetting of the baroreflex. Before pretreatment with atenolol, baseline AP and HR in WT mice (n = 5) were 104 ± 4 mmHg and 623 ± 28 beats/min, respectively. In the five ORX/ATX-Tg mice, basal AP and HR were 85 ± 5 mmHg and 651 ± 16 beats/min, respectively. After pretreatment with atenolol (5 mg/kg), AP and HR in WT mice were 100 ± 5 and 604 ± 31 beats/min, respectively. In ORX/ATX-Tg mice, AP and HR were 83 ± 5 mmHg and 609 ± 18 beats/min, respectively. AP in ORX/ATX-Tg mice was significantly lower than that in WT mice (P < 0.05, repeated-measures ANOVA followed by the post hoc comparisons of Student-Newman-Keuls procedure), irrespective of the presence of atenolol.

After pretreatment with atenolol, electrical stimulation to the PFA no longer elicited tachycardia but induced slight bradycardia, presumably through the baroreflex (Fig. 3, left). Although intravenous injection of phenylephrine alone induced a large decrease in HR (Figs. 3, middle and 4, left), simultaneous stimulation of the PFA with the injection of phenylephrine elicited only a small decrease in HR in the WT mice (Figs. 3, right and 4, middle) (46 ± 6% of the control, P < 0.001, repeated-measures ANOVA followed by the contrast test) showing resetting of the baroreflex. In a similar manner, injection of phenylephrine after chemical stimulation to the PFA elicited only a small decrease in HR (Fig. 4, right) (52 ± 11% of the control, P < 0.01). There was no difference in the changes in AP induced by phenylephrine among the three treatment groups (Fig. 4).


Figure 3
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  		Fig. 3. Typical example of inhibitory effect of PFA stimulation on baroreflex bradycardia in WT and Tg mice. Left: PFA was electrically stimulated for 30 s (PFA stim, horizontal bar) after pretreatment with atenolol (5 mg/kg ip). Middle: baroreflex bradycardia was induced by an intravenous injection of phenylephrine (PE; 50 µg/kg, arrow). Right: PE-induced bradycardia was reduced by simultaneous PFA stimulation in WT but not in Tg mice.

 

Figure 4
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  		Fig. 4. Effects of electrical or chemical stimulation of the PFA on PE-induced changes in arterial blood pressure ({Delta}AP) and heart rate ({Delta}HR) in WT and TG mice. Left: PE was injected without PFA stimulation (control). Middle: PE was injected during electrical stimulation of the PFA. Right: PE was injected ~5 min after chemical stimulation of the PFA by microinjection of bicuculline (1.0 mM, 20 nl) when the rise in AP was at maximum. Data are presented as means ± SE of 5 WT and 5 Tg mice. *P < 0.05 vs. WT mice; #P < 0.05 vs. control.

 
In ORX/ATX-Tg mice, phenylephrine induced a large decrease in HR (Figs. 3, middle and 4, left) that was comparable to the changes in WT, showing normally preserved baroreflex function in ORX/ATX-Tg mice at least under the control condition. However, attenuation of baroreflex that was seen in WT was never observed in ORX/ATX-Tg mice (Figs. 3, right and 4), either during electrical stimulation of the PFA (84 ± 7% of the control, P > 0.05) or after the injection of bicuculline to the PFA (88 ± 9% of the control, P > 0.05). Consequently, there was a significant difference between the changes in HR in ORX/ATX-Tg mice and those in WT mice during electrical stimulation of the PFA and after the injection of bicuculline to the PFA (Fig. 4, P < 0.05, repeated-measures ANOVA followed by the post hoc comparisons of Student-Newman-Keuls procedure).

Experiment 3: Baroreceptor Reflex in Conscious Animals at the Resting Condition

To confirm that the baroreflex function was normally preserved in ORX/ATX-Tg mice, we analyzed the baroreceptor reflex by the naturally occurring sequence method with AP and ECG recordings obtained from conscious animals in resting conditions. As expected, the slope of the regression line, i.e., gain of the baroreceptor reflex, and the baroreflex effectiveness index were not different between ORX/ATX-Tg and WT mice (Table 3). In addition, the number of the SAP ramps was comparable between the mutant and WT mice, showing that overall fluctuations in AP were similar between both genotypes.


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  		Table 3. Baroreflex parameters in ORX/ATX-Tg and wild-type mice evaluated by naturally occurring sequence method during conscious and resting conditions

 
Effect of Air Jet Stress

To exclude the possibility that the lower basal AP and the attenuation of the defense response in ORX/ATX-Tg mice in experiment 1 were due to a difference in sensitivity to the anesthetic used, we next examined the effect of air jet stress on AP and HR in conscious mice. During the resting condition before the air jet stress was applied, average AP in ORX/ATX-Tg mice (104 ± 2 mmHg, n = 8) was significantly lower than that in WT mice (122 ± 2 mmHg, n = 8, P < 0.05, Student’s t-test). Basal HRs of both genotypes were higher than those in experiment 1, in which cardiovascular parameters were measured under urethane anesthesia. Nevertheless, there was no difference in basal HR between ORX/ATX-Tg (657 ± 21 beats/min) and WT (653 ± 13 beats/min) mice, as was the case in experiment 1.

As expected, changes in AP and HR in response to air jet stress were smaller in ORX/ATX-Tg than in WT mice (Fig. 5, P < 0.01 for AP and P < 0.05 for HR, Student’s t-test), although the change of AP in WT mice was smaller than that after direct PFA stimulation in experiment 1.


Figure 5
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  		Fig. 5. Cardiovascular responses during air jet stress in conscious WT and Tg mice. Duration of air jet stress is indicated by the horizontal solid bar. Right: changes in AP and HR expressed as AUC during 5 min of the stress. Data are presented as means ± SE of 8 WT and 8 Tg mice. *P < 0.05, **P < 0.01 vs. WT mice.

 
Pharmacological Interventions

We have previously found that basal AP, but not HR, in orexin knockout mice was lower than that in the WT mice probably because sympathetic vasoconstrictor outflow was lower in orexin knockout mice (22). Since similar results were obtained in ORX/ATX-Tg mice (i.e., lower basal AP and similar basal HR compared with those in WT mice), we examined whether the same mechanism was applicable to ORX/ATX-Tg mice. Administration of a beta blocker, atenolol, slightly lowered the HR in both mutant (–13.7 ± 5.7%) and WT mice (–13.9 ± 3.3%) in a similar manner (P > 0.05, repeated-measures ANOVA followed by the post hoc comparisons of Student-Newman-Keuls procedure). The resultant AP was still lower in ORX/ATX-Tg than in WT mice (Fig. 6). On the other hand, prazosin lowered AP more in WT (–41.7 ± 3.9%) than in mutant mice (–29.1 ± 4.4%; P < 0.05). The resulting AP was not different between the two. There was no difference in HR between the two strains with any drugs used in the current experiment (Fig. 6).


Figure 6
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  		Fig. 6. Cardiovascular effects of intraperitoneal injection of atenolol and prazosin. Mean arterial pressure (A) and heart rate (B) were measured through indwelling catheters during pre- and postinjection periods in Tg mice (filled bars, n = 8) and in WT mice (open bars, n = 8).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
As a logical extension of our previous findings that orexin-deficient mice showed attenuation in some features of defense response, here we showed that other features of the defense response, namely, skeletal muscle vasodilation and suppression of reflex bradycardia were also blunted in the mutant mice. Taken together, we may say that orexin plays a role as a master switch to eliciting multiple features of the defense response that include increases in AP, HR, ventilation (Ref. 22 and present study), stress-induced analgesia (40), skeletal muscle vasodilation, and attenuation of baroreflex-induced bradycardia (present study). To our best knowledge, this is the first report showing a key role of a specific neuropeptide in complete elicitation of the defense response.

In this study, we used one transgenic line derived from the four founder lines, which was most extensively examined in our previous study (17). Positional effect of insertion may affect the localization, period, and/or strength of the expression of the transgene. Actually, the time course of the ablation depended on lines. After 12 wk of age, however, almost all orexin neurons were ablated in all lines (17). Because this animal model did not rely on the expression of the transgene per se but on the resultant ablation of neurons, we did not care about the amount of expression of the transgene. In addition, none of the founder lines showed ectopic expression of the transgene. Integration of the transgene should not have destroyed other gene loci, because we did not detect any neurological and histological abnormalities in the line we used in this study, as long as we maintain them as hemizygotes. In addition, there was no difference in phenotype among the four founders when evaluated by EEG/EMG recording, histological examination, and observation of behavioral arrest after 12 wk of age (17). Therefore, it is plausible to suppose the same results would be obtained if we examined another transgenic line.

In each experiment of the current study, we used different sets of animals that differed in age and/or number of backcrossings to C57BL/6 due to availability of the animals (Table 1). It is noteworthy that there was no difference in body weight between ORX/ATX-Tg and WT mice because the original report by Hara et al. (17) showed an obesity phenotype in ORX/ATX-Tg mice with mixed (75% C57BL/6–25% DBA2) genetic background after the age of 12 wk. A recent report (18) showed, however, no such phenotype in animals with a nearly pure C57BL/6 background (<0.1% DBA2) when they were fed with normal chow. The latter mice were obtained by backcrossing the former with C57BL/6 eight times (N8). Because N5 (0.8% DBA2) and N6 (0.4% DBA2) generations of the mice were used in the current experiment, the mixture of >0.8% DBA2 background might have affected the obesity phenotype of the founder mice.

It should be noted that the baroreflex parameters at the basal condition in conscious animals were not different between ORX/ATX-Tg and WT mice (Table 3). In addition, after the administration of atenolol without PFA stimulation in the anesthetized condition, reflex bradycardia in response to similar changes in AP was not different between ORX/ATX-Tg and WT mice (Fig. 3, left). We recognize that the baroreflex gain provided by the sequence method concerns only a small range of AP. We only examined pressor-induced bradycardia in the anesthetized animals of the present study. However, ORX-KO mice did not show any abnormality in both pressor-induced bradycardia and depressor-induced tachycardia when AP was changed in a wide range (Y. Kayaba and T. Kuwaki, unpublished observation). On the other hand, immunohistochemical studies suggested orexinergic innervation in the nucleus tractus solitarius, dorsal motor nucleus of vagus, and nucleus ambiguus (7, 8, 37). Although no electrophysiological evidence showing monosynaptic projection is available at present, it is likely to be a direct projection because orexin-containing cell bodies are exclusively located in the hypothalamus. A whole cell patch-clamp study showed that cardiac vagal neurons in the nucleus ambiguus were excited by the application of orexin (9). Our data indicated that these orexinergic innervations seemed not to participate in the tonic control of the baroreceptor reflex at the basal condition but might be activated on demand during reactions, such as the defense response, to play a modulatory role.

In our previous study using ORX-KO mice (22), we found that increases in AP, HR, and ventilation during the defense response were significantly attenuated but not completely abolished. Therefore, we suspected possible involvement of other neuropeptides or modulatory factors contained in the orexinergic neurons. These were dynorphin (6), galanin (16), glutamate (1, 32), and nitric oxide (5). These substances are considered deficient in ORX/ATX-Tg mice because almost all of the orexin-containing neurons were ablated in this mutant (17). In fact, expression of dynorphin in the hypothalamus is abundant in WT and ORX-KO mice but completely absent in ORX/ATX-Tg mice (6). Contrary to our expectation, however, attenuation of the defense response in ORX/ATX-Tg mice was not exaggerated but similar to that observed in ORX-KO mice. Response magnitudes to bicuculline were similar in both mutants when increases in AP, HR, and ventilation were recalculated as the relative value to the average of the corresponding WT (Table 4). Moreover, a lower basal AP in ORX/ATX-Tg mice (~20 mmHg) was similar to that observed in ORX-KO mice (22). Both ORX/ATX-Tg and ORX-KO mice showed attenuated AP and HR responses to natural stimuli in the conscious condition, although quantitative comparison was difficult in this case because of the different experimental setups (air jet stress in ORX/ATX-Tg and resident-intruder test in ORX-KO mice). Therefore, the lack of orexin seemed to be responsible for the attenuated defense response (at least the rise in blood pressure, HR, and respiratory minute volume) and low basal AP, and contributions of other neuropeptides or modulatory factors in the orexinergic neuron seemed to be minor. Contributions of other neuropeptides or modulatory factors in vasodilation of the skeletal muscle and the shift of baroreflex remains an open question because we have not tested these parameters in ORX-KO mice.


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  		Table 4. Magnitude of the bicuculline-induced defense responses expressed as the relative value to the average of the corresponding wild-type mice

 
In both ORX/ATX-Tg and ORX-KO mice, increases in AP, HR, and ventilation were not completely abolished but were about a half of those in the corresponding WT with 1.0 mM of bicuculline (Table 4). This observation may indicate contribution of other transmitter(s) that are distributed nearby but outside of the orexin-containing neurons in the defense response. Further studies are needed to clarify this issue.

Several reports showed that stress activates orexinergic neurons (14, 20, 40, 42, 45). Numerous neurons in the amygdala, a putative center for fear, anxiety, and biological value judgment (31), are retrogradely labeled by transsynaptic transport of tetanus toxin expressed by the orexin promoter (34). Moreover, orexinergic neurons in the lateral hypothalamic area and the PFA send axon collaterals to the primary motor cortex and the cardiosympathetic outflow system (24). These reports are in line with our hypothesis that orexin contributes to the defense response.

Orexinergic neurons projected to the cardiovascular centers (2, 9, 12, 15, 37) and respiratory centers (39, 43) in the medulla oblongata and the spinal cord. Some of these nuclei may be involved in the elicitation of each feature of the defense response examined here. Moreover, orexinergic neurons innervated sympathetic premotor neurons controlling brown adipose tissue (3, 30). Therefore, stress-induced hyperthermia (27) may also be controlled by orexin.

As was the case in ORX-KO, the cause of low basal AP in ORX/ATX-Tg mice seemed to be lower in sympathetic vasoconstrictor outflow, and cardiac sympathetic outflow seemed normal (Fig. 6). In this regard, it is of interest to note that fight-flight (active) type, but not defeated inactive type of repeated stress, induced hypertension in rats (19). Although the reverse is not necessarily true, there may be a link between the defense response (fight or flight response) and basal AP.

In conclusion, this study provided further support for our hypothesis that orexin-containing neurons in the PFA play a role as a master switch to activate multiple efferent pathways of the defense response and also operate as a regulator of basal AP. Orexin, but not other neuropeptides or modulatory factors contained in the neurons, seemed to be important for these roles.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Part of the study was supported by Grants-in-Aid for Scientific Research (16590162, 17590183) from the Ministry of Education, Culture, Sports, Science and Technology, Japan and grants from the Smoking Research Foundation and Mitsui Life Social Welfare Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Megumi Shimoyama for help in editing the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kuwaki, Dept. of Molecular and Integrative Physiology, Chiba Univ. Graduate School of Medicine, 1-8-1 Chuo-ku, Chiba 260-8670, Japan (e-mail: kuwaki{at}faculty.chiba-u.jp)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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