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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota
Submitted 9 July 2007 ; accepted in final form 5 September 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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limb-girdle muscular dystrophy; euthyroid sick syndrome; hypothyroid
- and 
-sarcoglycan genes also results in the loss of the sarcoglycan-sarcospan, dystroglycan complexes, sarcolemmal neural nitric oxide synthase, and the 
-sarcoglycan-containing complex in skeletal muscle (11, 12, 53).
The BIO 14.6 hamster is an animal model of limb-girdle muscular dystrophy due to a mutation of -sarcoglycan gene, resulting in the absence of the sarcoglycan protein and reduced expression of 
-dystroglycan (24). In addition to limb-girdle muscular dystrophy, BIO 14.6 hamsters exhibit low triiodothyronine (T3 levels) but normal thyroxine levels resulting in euthyroid sick syndrome (49, 51) and later in life develop cardiomyopathy (10).
Thyroid hormone status may affect breathing in a variety of ways including changing the structure and function of the diaphragm, altering the levels of neurotransmitters or receptors, and regulating the function of central nervous system channels (4, 8, 21, 22). BIO 14.6 dystrophic hamsters exhibit lower ventilatory responses to hypoxia and hypercapnia, as well as lower O2 consumption, and CO2 production relative to the control hamsters (48). Previous research indicates that golden Syrian hamsters made hypothyroid by the administration of propylthiouracil (PTU) also exhibit lower ventilatory responses to hypoxia compared with euthyroid hamsters (50). Moreover, thyroxine supplementation in dystrophic hamsters was able to normalize their ventilatory responses to hypoxia (49). Thus, thyroid status affects control of breathing in hamsters.
In awake and sleeping human subjects, hypothyroidism influences control of breathing (16, 38, 46, 55). In addition, hypothyroidism can cause obstructive sleep apnea (42, 39, 52). Following thyroxine treatment ventilation improved greatly in hypothyroid patients with sleep apnea (27). Hypothyroidism may decrease ventilatory neural output and may affect the expression of a number of neurotransmitters and their receptors including serotonin (9, 14, 30, 36, 47).
Exposure to intermittent hypoxia lasting a few minutes can induce a form of neuronal plasticity termed ventilatory long-term facilitation (LTF) (1). Studies involving patients with obstructive sleep apnea and other studies in rats, reported that when the subjects were exposed to air or hyperoxia following exposure to several bouts of intermittent hypoxia, ventilation and neural activity from phrenic, vagal, intercostals, and hypoglossal motoneurons increased above baseline values, thus, exhibiting LTF (9, 35). LTF is dependent upon activation of serotonin receptors (especially 5-HT2A receptors), elevated levels of brain-derived neurotrophic factor in the central nervous system, (2, 31, 34) affecting catecholamines (26), activation of dopamine D2 receptors (Schlenker EH, unpublished observations). LTF in humans, as well as animals, can also be affected by arousal state (awake or sleeping), age, gender, and sex steroid hormones (31, 35, 54). Thus, intermittent hypoxia may not always induce LTF. The role of thyroid hormones in eliciting ventilatory LTF is not known.
Research in our lab indicated that dystrophic BIO 14.6 hamsters exhibited long-term depression (LTD) following intermittent hypoxia (Schlenker EH, unpublished observation). Therefore, low thyroid levels may contribute to the development of LTD. The mechanism by which LTD develops in low T3 syndrome is not currently known, but may involve thyroid hormone modulation of serotoninergic and catecholaminergic function and effects on intracellular signaling systems.
In the present study, ventilation of euthyroid golden Syrian, BIO 14.6 (dystrophic), and PTU-treated (hypothyroid) hamsters was recorded at baseline, during exposure to intermittent hypoxia, and following intermittent hypoxia after administration of vehicle or 5-HT2A receptor antagonist, MDL. We first hypothesized that ventilation in air and during intermittent hypoxia would be lower in the dystrophic and PTU-treated hamsters relative to that in euthyroid hamsters and depressed by MDL treatment only in the euthyroid group. Secondly, we hypothesized that following intermittent hypoxia the euthyroid group would exhibit ventilatory LTF, the dystrophic group would exhibit LTD, and ventilation in the PTU-treated group would be similar to the dystrophic group. Finally, we hypothesized that MDL should cause LTD in the euthyroid group and have no effect on ventilation following exposure to acute intermittent hypoxia in the dystrophic and PTU-treated hamsters.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Twenty male golden Syrian hamsters aged 74 days obtained from Harlan Sprague Dawley, Indianapolis, IN and 11 male dystrophic hamsters aged 76 days (strain BIO 14.6) obtained from BioBreeders, Watertown, MA were used in this study. Eleven Syrian hamsters served as the euthyroid group, while nine hamsters treated with propylthiouracil served as the hypothyroid group. Before and after experimental procedures animals were housed in Ancare cages (22.9 x 43.2 cm) with a microisolator top. Bedding consisted of Aspen chips. Animals were housed in groups of two or three with a 14:10-h light-dark cycle. Food (8604 rodent pellets obtained from Harlan Sprague Dawley) and water or 0.01% propylthiouracil (to induced hypothyroidism) were available ad libitum (50) Three days before experimental procedures commenced, animals were acclimated to the respiratory-metabolic chamber and experimental procedures. The University of South Dakota Animal Care and Use Committee approved all methods used in this experiment.
Respiratory Metabolic Chamber
Respiratory and metabolic measurements were made in a 20.2 x 7.9 cm Plexiglas cylinder. One end of the cylinder was closed and contained openings for calibration (using a 1 ml glass syringe), measuring chamber temperature (using a Taylor series 9940 thermometer), determining pressure changes within the chamber associated with breathing using a Statham low pressure transducer (coupled to an Acknowledge System by BioPac data acquisition system), and an input port for air or 10% oxygen in nitrogen. The other end of the cylinder was closed and contained two openings. One opening was used to determine flow rate through the chamber using a Gilmont rotameter. The other opening could be connected to a "leak" or connected to a gas analyzer (Vacu-Med model 17515A gas analyzer) used to measure the fractional content of CO2.
Tidal volume was evaluated using the method of Schlenker (48). Inspiratory and expiratory times were measured from 10 breaths and were averaged for each portion of the experiment. Frequency was determined by dividing 60 s per min by the total of inspiratory plus expiratory time. Minute ventilation was calculated by multiplying tidal volume by frequency. CO2 production was determined by subtracting the fractional content of CO2 in the input air from the fractional content of CO2 in the output air and multiplying the difference by the flow rate. The ventilatory equivalent, a measure of how well ventilation is matched with metabolism, was calculated by dividing minute ventilation by CO2 production. Flow rates through the chamber averaged 680 ml/s. Barometric pressure was measured with a W. M. Welch Scientific Company barometer and averaged 720 Torr.
Protocol
On the day of the experiment a hamster was weighed and injected with either the vehicle (5% dimethyl sulfoxide in saline) or the 0.5 mg/kg of the serotonin (5-HT2A) receptor antagonist, MDL 100,907 (generously donated by Sanofi-Aventis, France) in vehicle. The hamster was then placed in the chamber for 30 min of acclimatization to air and to ensure that the MDL would be taken up into the central nervous system (25, 28). At the end of the acclimatization period ventilatory parameters, flow rate, and CO2 content were measured.
Subsequently, animals were exposed to five bouts of 5 min each of air interspersed with 5 min of 10% O2 in nitrogen, which constituted the intermittent hypoxic exposure (34). Frequency, tidal volume, and minute ventilation were evaluated after each intermittent exposure to hypoxia (H1, H2, H3, H4, and H5). Moreover, after H5, CO2 production was determined and ventilatory equivalent was calculated.
While the hamsters breathed air following the intermittent hypoxic exposure, measurements were made in 15-min increments (A15, A30, A45, A60 min) (37). Frequency, tidal volume, minute ventilation, and CO2 production were determined and ventilatory equivalent was calculated after 15, 45, and 60 min following hypoxia. Frequency, tidal volume, and minute ventilation were also measured after 30 min posthypoxia. After the hamster was removed from the chamber, its body temperature was measured by using a Sensortek BAT-12 thermometer with a Physiotemp thermocouple. Each animal received two treatments: vehicle and MDL. The time period between vehicle and MDL treatments for each animal was <7 days.
Tidal volume, minute ventilation, and CO2 production were corrected for body weight (variable/1,000/body wt). Statistical analysis included a one-way analysis of variance to compare baseline values and body temperatures and a repeated two-way ANOVA was used to determine effects of group (control, dystrophic, and PTU) and treatment (vehicle and MDL) on body temperature, CO2 production, and ventilatory parameters. If the analysis was significant (P < 0.05), post hoc tests using Student-Newman-Keuls and Holms t-tests, corrected for multiple comparisons, were used to evaluate the results. Data were analyzed using the StatMost program (Dataxiom Software, Los Angeles, CA). Significance for all experiments was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Body weight in the PTU-treated group (132 ± 8 g) was significantly higher than that of the euthyroid (123 ± 10 g) and dystrophic groups (112 ± 10 g, P = 0.0010). There was not a difference in body weights among the three groups of hamsters comparing vehicle and MDL treatment (data not shown).
Body temperature. With vehicle treatment, the dystrophic (35.2 ± 0.3°C) and PTU-treated groups (35.3 ± 0.5°C) had significantly higher body temperatures than did the euthyroid group (34.5 ± 0.3°C, P = 0.0075). Following MDL treatment, the group differences were no longer present. In fact, body temperature decreased in all groups with MDL treatment relative to vehicle: 0.5°C in the euthyroid group, 0.9°C in the dystrophic group, and 0.7°C in the PTU-treated group (P < 0.0001).
Comparison of Respiratory and Metabolic Parameter: Vehicle Treatment
Our first hypothesis was that ventilation in air would be lower in the dystrophic and PTU-treated hamsters relative to that in euthyroid hamsters. Looking at the individual components that make up ventilation, frequency and tidal volume, we determined that frequency was significantly higher in the PTU-treated group relative to the dystrophic group (P = 0.028) (Table 1). No differences were observed in frequency between the euthyroid and PTU-treated groups or between the euthyroid and dystrophic groups. Body weight-corrected tidal volume was significantly lower in the PTU-treated group relative to the control group (P = 0.0463) (Fig. 1A), but similar to that of the dystrophic group. Body weight-corrected minute ventilation was significantly lower in the dystrophic group relative to the control group (P = 0.0275) (Fig. 2A), but comparable between the control and PTU-treated groups. CO2 production, corrected by body weight, was significantly lower in the dystrophic and PTU-treated groups relative to the control group (P = 0.002) (Fig. 3A). There were no differences in ventilatory equivalents among the three groups (Fig. 4A).
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We hypothesized that MDL would depress ventilation in the euthyroid, but not dystrophic and PTU-treated groups. Relative to vehicle, MDL treatment affected most parameters differently in the three groups. MDL treatment relative to vehicle increased frequency in the euthyroid group (P = 0.0136) (Table 1), but had no effect in the dystrophic or PTU-treated groups. Tidal volume was significantly lower in the euthyroid (P = 0.028) and PTU-treated (P = 0.002) groups following MDL treatment relative to vehicle (Fig. 1A relative to Fig. 1B). In contrast, MDL treatment did not affect tidal volume in the dystrophic group. Minute ventilation was significantly lower in both the euthyroid (P = 0.009) and PTU-treated (P = 0.0287) groups relative to vehicle, but MDL treatment did not affect minute ventilation in the dystrophic group (Fig. 2A relative to Fig. 2B). CO2 production was significantly higher in the MDL-treated euthyroid group relative to vehicle treatment (P = 0.007, Fig. 3A relative to Fig. 3B), but no effect of MDL treatment was noted in the dystrophic and PTU-treated groups. Only in the euthyroid group was the ventilatory equivalent significantly lower with MDL treatment relative to vehicle (Fig. 4A vs. Fig. 4B; P = 0.0325).
In summary, during exposure to air, MDL decreased body temperature in all groups. In the dystrophic group, MDL had no effect on respiratory and metabolic parameters, whereas in the euthyroid group all variables were affected by MDL treatment. In the PTU-treated group, the effects of MDL treatment on respiratory and metabolic parameters fell somewhere between those of dystrophic and euthyroid groups.
Metabolic and Ventilatory Responses to Intermittent Hypoxia: Vehicle Treatment
We hypothesized that during intermittent hypoxia, ventilation would be lower in the dystrophic and PTU-treated groups relative to the euthyroid group. Vehicle-treated hamsters in all three groups when exposed to intermittent hypoxia did not increase frequency relative to baseline values (Table 1). Group differences observed at baseline were maintained throughout exposure to intermittent hypoxia. In contrast, intermittent hypoxia had more complex effects on body weight-corrected tidal volume in the three groups relative to baseline. With vehicle treatment, tidal volume increased during exposure to intermittent hypoxia in all groups relative to baseline. Ratios of tidal volume during hypoxic exposure to tidal volume at baseline were not different among groups with vehicle treatment (Table 2).
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With vehicle treatment, repeated exposures to hypoxia showed that body weight-corrected minute ventilation was significantly lower in the dystrophic and PTU-treated groups relative to the euthyroid group (Fig. 2A). CO2 production was not affected by exposure to intermittent hypoxia relative to baseline in any group (Fig. 3A). In the euthyroid group, CO2 production was higher relative to the dystrophic and PTU-treated groups, but there are no differences between the dystrophic and PTU-treated groups. With vehicle treatment exposure to intermittent hypoxia had no effect on ventilatory equivalent relative to baseline values in any group (Fig. 4A).
Metabolic and Ventilatory Responses to Intermittent Hypoxia: MDL Treatment
We hypothesized that MDL would depress ventilation during intermittent hypoxia in the euthyroid, but not dystrophic and PTU-treated groups. After MDL treatment, body weight corrected tidal volume in the PTU-treated group was lower than in the euthyroid and dystrophic groups (Fig. 1B). Moreover, there was no significant difference between control and dystrophic values from H1 to H5. With MDL treatment tidal volume ratios (value during hypoxic exposure/baseline value) in the euthyroid and PTU-treated groups decreased relative to vehicle ratios (Table 2). Moreover, with MDL treatment, tidal volume ratios of each hypoxic exposure relative to baseline values decreased from H1 to H5 in the euthyroid (P = 0.0067), but not in the dystrophic and PTU-treated groups (Table 2), and the tidal volume, ratios during intermittent hypoxic exposures were lower in the dystrophic group relative to the dystrophic and PTU-treated groups (F2,64 = 3.89, P = 0.042; Table 2). All groups increased minute ventilation at H1 relative to baseline with MDL treatment (Fig. 2B).
During exposure to intermittent hypoxia after MDL treatment relative to vehicle, minute ventilation was lower (P < 0.02) in the PTU and dystrophic groups relative to euthyroid hamsters at H4. During repeated exposures to hypoxia, the ratios of minute ventilation responses relative to baseline decreased in the dystrophic (P = 0.0004), PTU-treated (P = 0.0021), and control groups (P = 0.0005) (Table 3).
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With MDL treatment, ventilatory equivalents were decreased in the euthyroid group relative to the dystrophic and PTU-treated groups (F1,32 = 21.82, P < 0.0001, Fig. 4B). Overall, there was a decrease in ventilatory equivalent in the control group with MDL treatment relative to vehicle treatment due to a decrease in minute ventilation and an increase of CO2 production.
Respiratory and Metabolic Responses Following Intermittent Hypoxia Vehicle Treatment
We hypothesized that following intermittent hypoxia the euthyroid group would exhibit ventilatory LTF, the dystrophic group would exhibit LTD, and ventilation in the PTU-treated group would be similar to the dystrophic group. Following intermittent hypoxia compared with baseline values there was no effect on frequency in the vehicle-treated euthyroid group, but frequency decreased in the dystrophic (P = 0.0039) and PTU-treated (P = 0.0007) groups (Table 1). However, following intermittent hypoxia, tidal volumes in the dystrophic and PTU-treated groups were lower than those in the control group at A15 (P = 0.0005), A30 (P = 0.0153), and A60 (P = 0.0013) (Fig. 5A).
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Respiratory and Metabolic Responses Following Intermittent Hypoxia MDL Treatment
We had hypothesized that MDL treatment would eliminate ventilatory LTF in the euthyroid hamsters, but have no effect on the dystrophic and PTU-treated groups. Although we did not report LTF with vehicle treatment in the euthyroid group, MDL did depress ventilation in that group following intermittent hypoxia. The mechanisms for effects of MDL on ventilation are described below.
Following MDL treatment, frequency decreased in the euthyroid group after intermittent hypoxia relative to baseline (P = 0.005) and in the dystrophic (P = 0.0183) and the PTU-treated (P = 0.0044) groups relative to baseline values (Table 1). Thus, with MDL treatment the pattern of frequency response of the euthyroid group became similar to that of the dystrophic and PTU-treated groups.
With MDL relative to vehicle treatment following exposure to intermittent hypoxia, the euthyroid group decreased tidal volume (F1,45 = 137, P < 0.0001), which remained constant from A15 to A60, and the PTU-treated group also decreased tidal volume (Fig. 5A vs. 5B; P < 0.0001). In contrast, with MDL treatment tidal volumes in the dystrophic group were higher than that of the euthyroid and PTU-treated groups at A15 (P < 0.0001), A30 (P < 0.0001), and A45 (P = 0.0002), and higher than those of the PTU-treated group at A60 (P < 0.0001) (Fig. 5B).
With MDL treatment, following exposure to intermittent hypoxia relative to baseline there was a significant decrease of minute ventilation in the euthyroid group at A15 (P = 0.0396) and A30 (P = 0.0104) (Fig. 6B). MDL treatment had no significant effects on minute ventilation in the dystrophic group following intermittent hypoxia, and ventilation decreased significantly in the PTU-treated group only at A60 (P = 0.0391). Thus, MDL treatment depressed ventilation following intermittent hypoxia in the control hamsters.
With MDL relative to vehicle treatment, CO2 production increased in the euthyroid group (P < 0.0001) (Fig. 3B). CO2 production increased slightly in the dystrophic group at A60 (P = 0.0461) with MDL treatment (Fig. 3B). In the PTU-treated group there was no significant effect on CO2 production with MDL treatment.
Following intermittent hypoxia, MDL treatment relative to vehicle decreased ventilatory equivalent in the euthyroid group, but not in the other two groups (P < 0.0001) (Fig. 6B). Moreover, with MDL treatment the ventilatory equivalents at A15, A45, and A60 were higher in the PTU and dystrophic groups relative to those in the control group. Thus, MDL treatment resulted in a marked decrease in ventilatory equivalent only in the euthyroid group, due primarily to a large increase in CO2 production and a decrease in minute ventilation.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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When comparing MDL treatment to vehicle, ventilation was decreased in the euthyroid group at baseline, during, and following intermittent hypoxia; effects not observed in the dystrophic group. CO2 production was increased in the euthyroid group with MDL treatment relative to vehicle resulting in a ventilatory equivalent that was significantly lower with MDL relative to vehicle treatment. Finally, with MDL treatment, body temperature was significantly lower in all groups relative to vehicle. The subsequent sections discuss the relevancy of these findings.
Body temperature. MDL treatment decreased body temperature in all groups. Body temperature is the balance between heat production and heat loss. Factors contributing to heat production include increasing sympathetic nervous system activity that increases metabolic rate and brown fat activity (20). Vasomotor tone (dilation and constriction) can affect body temperature. Heat loss occurs via vasodilatation and may also be lost through an increase in ventilation (20). Serotonin and its receptors can modulate the function of the sympathetic nervous system, and thereby affect body temperature. For example, the regulation of sympathetic nerves in a cutaneous bed by 5-HT receptors was investigated in rabbits and rats (5) in which blood flow was measured by implanted Doppler ultrasonic flow probes and body temperature was measured by telemetric probes. The effects of (+/–)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a specific 5-HT2A receptor agonist, were determined on cutaneous blood flow (ear pinna in rabbits, tail in rats), mesenteric blood flow, and arterial pressure. Hyperthermia induced by DOI was preceded by reduced blood flow to the cutaneous bed, with no change in the flow to the mesenteric bed. In rabbits, the 5-HT2A receptor antagonists (ketanserin and AC90179) reversed the ear pinna vasoconstriction induced by DOI (5), whereas in rats, ketanserin reversed tail DOI-induced vasoconstriction and the hyperthermia. Thus, 5-HT2A receptor-associated vasoconstriction of the cutaneous bed was mediated by the sympathetic nervous system can result in hyperthermia that is reversed by 5-HT2A antagonists.
Several other studies have shown similar results in that stimulation of 5-HT2A receptors by specific agonists cause hyperthermia in laboratory animals (3, 32, 37). Currie et al. (13) evaluated the role of neuropeptide Y (NPY) on 5-HT receptors in relation to food intake and energy metabolism in rats. Specific 5-HT agonists or antagonists were administered 5 min before paraventricular nucleus injection of NPY, and oxygen consumption, carbon dioxide production, and respiratory quotient were measured 2 h postinjection. Results suggested that 5-HT2A receptors, within the medial paraventricular nucleus, modulated the action of NPY on food ingestion and energy metabolism (15).
Chueh et al. (7) investigated the effects of the serotoninergic system on thermal effects of puerarin, a 5-HT1A receptor agonist, microinjected into the third cerebral ventricle or given systemically. Colonic temperature in rats was measured every minute. The study reported that the intraperitoneal administration of puerarin elicited hypothermia (9). In addition, the systemic or central administration of puerarin caused a decrease in colonic temperature and hypothalamic 5-HT efflux. The 5-HT2A receptor antagonist, ketanserin, had additive hypothermic effects with puerarin. Researchers concluded that puerarin exerts hypothermic and antipyretic effects by activating 5-HT1A receptors and/or antagonizing 5-HT2A receptors in the hypothalamus. Previous research has suggested that the 5-HT2A receptor agonist, D-lysergic acid diethylamide and the 5-HT2A/2C receptor agonist DOI elicit hyperthermia in rats and rabbits (40). Also, 5-HT2A receptor antagonists reversed hyperthermia caused by DOI in rats (41, 44).
Although our hypotheses did not include effects of MDL on body temperature, MDL treatment significantly decreased body temperature in all groups. Since MDL increased CO2 production in the control group, the decrease in body temperature with MDL treatment may have occurred through vasodilatation or a shift of blood from the core to the periphery. Further studies are needed to determine the mechanisms responsible for MDL decreasing body temperature in hamsters.
Comparison of Ventilation in Euthyroid, Hypothyroid and Dystrophic Hamsters
The lower ventilation in air, during and after intermittent hypoxia of dystrophic hamsters noted in the present study concurs with previous findings from our laboratory (48, 49, and Schlenker EH, unpublished observations). Unlike the elaboration of ventilatory LTF noted in hamsters in a previous study (Schlenker EH, unpublished results), hamsters in the present study did not exhibit LTF. The factors responsible for the different findings are not known but not all studies using intermittent hypoxia protocols elicited LTF in rats or humans (35, and references therein). Factors such as the sleep state of a subject may determine whether LTF could be elicited following exposure to intermittent hypoxia. Hamsters in the present study were studied during the light cycle which corresponds to their "sleep periods." We did not instrument the hamsters to evaluate their arousal state, but made measurements when the animals’ eyes were open and they were resting as we did in the previous study. One factor that may differ is the time of the year the studies were conducted. Ventilatory LTF was induced in hamsters studied in the late fall and winter in pilot studies, whereas the present study was conducted in the summer. Additional investigations needed to be conducted to determine whether seasonality may affect the production of ventilatory LTF in the hamster.
Serotoninergic receptors and control of breathing. The results of the present study in euthyroid hamsters suggest that 5-HT2A receptors may be involved in the ventilation in air and ventilatory responses to hypoxia. In the present study, MDL (a selective 5-HT2A receptor antagonist) decreased minute ventilation relative to vehicle in the euthyroid, but not in dystrophic hamsters exposed air and repeated intermittent hypoxic exposures.
The serotoninergic system has been implicated in modulating ventilation in air in response to hypoxia (45). Herman et al. (23) examined the effects of the combination of a selective 5-HT1A receptor agonist, tetralin [8-hydroxy-(2-di-n-propylamino)] and ketanserin (a 5-HT2A/2C receptor antagonist) on the ventilatory acclimatization to hypoxia in conscious goats. The combination of the two receptors increased minute ventilation, attributed to an increased tidal volume as frequency did not change with hypoxic exposure relative to air exposure. The researchers concluded that 5-HT receptors may affect ventilatory responses to acute hypoxia exposure.
Ventilation following intermittent hypoxia may increase above baseline values. This increase is termed LTF, and there is evidence that serotoninergic receptors help modulate this effect. Kinkead and Mitchell (29) examined the effects of 5-HT2 antagonists on ventilatory LTF in rats using ketanserin (a 5-HT2A/2C receptor antagonist). Phrenic and hypoglossal nerve responses were measured at baseline and following hypoxic exposure in paralyzed, artificially ventilated rats pretreated with ketanserin. Rats were exposed to three 5-min bouts of hypoxia, separated by 5 min of hyperoxia. During air exposure at baseline, ketanserin had no effect on phrenic and hypoglossal responses relative to saline treatment. However, during exposure of rats to hypoxia, ketanserin decreased phrenic, but not hypoglossal burst amplitude. Ketanserin augmented the frequency decline following hypoxic exposure relative to that of saline-treated rats. Thus, these results suggested that 5-HT2A/2C receptor activation affects short-term phrenic responses to intermittent hypoxia and is necessary in rats for LTF.
Although euthyroid hamsters in the present study did not exhibit LTF after vehicle treatment, MDL (acting to block 5-HT2A receptors) caused ventilatory LTD in this group, but not in the dystrophic or PTU-treated groups, suggesting that alternations in thyroid status may affect 5-HT2A receptor modulation of breathing following intermittent hypoxia. From a mechanistic perspective, blocking the production of LTF with a 5-HT2A receptor antagonist may not be the same as depressing ventilation following intermittent hypoxic exposure when LTF is not produced as noted in the present study. Perhaps in hamsters, hypoxia causes release of inhibitory neurotransmitters such as 
-amino-butyric acid (GABA; 14) that could counteract the excitatory effects of 5-HT during intermittent hypoxia and thereby preventing production of LTF. After administration of MDL, the effects of the inhibitory neurotransmitter may predominate and lead to depression of ventilation following exposure to intermittent hypoxia.
Hypothyroidism and serotoninergic function. Several studies have linked hypothyroidism and altered serotoninergic receptor function. Ostuni et al. (43) determined that thyroid hormone status affected 5-HT-stimulated secretion of parotid amylase in rats. The effect of 5-HT on amylase secretion was examined in a control (euthyroid) group, a hypothyroid group (made hypothyroid by a surgical thyroidectomy), and a hyperthyroid group (made hyperthyroid by the administration of sodium l-T3). At baseline, hyperthyroid rats released significantly more amylase relative to the control rats. After administration of 5-HT, amylase release was significantly lower in the hypothyroid group and higher in the hyperthyroid group relative to the control group (43). In all three groups, the effects of 5-HT were significantly blocked by the addition of methysergide, a nonspecific 5-HT receptor antagonist. However, ketanserin had no effect on amylase release in the control or hypothyroid animals and only partially blocked the effect of 5-HT in the hyperthyroid rats. Thus, researchers concluded that 5-HT induces amylase release acting on specific serotoninergic receptors and that thyroid levels modify the effect of 5-HT.
Kulikov and Jeanningro (30) evaluated the effects of hypothyroidism on 5-HT1A and 5-HT2A receptors in rat brains. Thyroidectomized rats were divided into two groups: one group received an iodine-free diet while the other group received thyroxine treatment. Saline-treated rats served as the control (euthyroid) group. There was a significant decrease in [3H]ketanserin binding in the frontal cortex of the hypothyroid rats relative to the control rats; the decrease was reversed with thyroxine treatment. Binding of [3H]8-OH-DPAT (5-HT1A receptor antagonist) in the midbrain was similar in both hypothyroid and control rats (33).
Rastogi and Singhal (47) examined the effect of hypothyroidism and thyroid hormone replacement on serotoninergic neurons in young rats and consequences of thyroid status on development. Hypothyroidism was induced with a single intraperitoneal injection of 200 µCi of 131I in one-day-old rats. Then, L-T3 at a dose of 10 µg/100 g was injected daily for 25 days beginning 5 or 120 days following 131I treatment. Midbrain tryptophan hydroxylase (the enzyme involved in the production of serotonin) activity was significantly decreased (24%) 30 days following administration of 131I at day 1 (47). The investigators found that lack of thyroid hormone early in development decreased 5-HT levels in the striatum, midbrain, and cerebellum. T3 treatment beginning 5 days after 131I treatment enhanced tryptophan hydroxylase activity (by 34%), as well as tryptophan levels (by 35%) (47). Administration of T3 120 days after I31 treatment did not have a significant effect on tryptophan hydroxylase activity. These results suggest that during a critical period in early life, thyroid hormones are needed for normal development of 5-HT system in the brain.
We noted a relationship between hypothyroidism and the 5-HT2A receptor function in the present study. CO2 production was significantly lower in both the dystrophic and PTU-treated groups relative to the control group with MDL treatment at baseline and during exposure to intermittent hypoxia. Ventilatory equivalent was significantly higher in both the dystrophic and PTU-treated groups relative to the control group with MDL treatment at baseline and during exposure to intermittent hypoxia. With MDL treatment, tidal volume in the dystrophic group was higher than that of the control and PTU-treated groups at A15, A30, and A45. Moreover, with MDL treatment the dystrophic group's tidal volume was higher than that of the PTU-treated group at A60. These results support the previous research cited above suggesting there is a relationship between hypothyroidism, 5-HT2A receptors, and control of breathing. However, MDL did not have the same effect in the dystrophic group as it did in the PTU-treated group, especially with tidal volume following hypoxia, suggesting that the underlying mechanisms may be different between the groups and negate, in part, our hypothesis that 5-HT2A function is similar in these two groups.
Potential Mechanisms for Ventilatory LTD Production in Hypothyroidism
A potential mechanism by which hypothyroidism may interact with serotonin to produce LTD is related to a model proposed by Mahamed and Mitchell (35) for production of LTF. They proposed that hypoxia causes release of serotonin from raphé neurons that acts on phrenic neuron 5-HT2A receptors and initiates a signaling cascade that produces brain-derived neurotrophic factor. By binding to its receptor tyrosine protein kinase receptor, trkB, the interaction results in phosphorylating protein kinase B (pAkT) and/or extracellular-regulated kinases that then phosphorylate glutamatergic subunits resulting in insertion of AMPA and or NMDA receptors postsynaptically, ultimately leading to LTF.
Thyroid hormones can regulate the phosphorylation of Akt and in that way possibly modify the production of LTF. For example, hypothyroid rats show lower levels of pAkt in hearts than do euthyroid animals (Gerdes AM and Lui Y, unpublished observations). Administration of T3 normalized pAkt levels. Thus, if hypoxia causes release of GABA and serotonin in the hypothyroid hamster and the pAkt levels do not increase and/or there are fewer 5-HT2A receptors, LTD should result.
Study Limitations
In this study, we used only one dose of MDL that had profound effects on ventilatory and metabolic parameters in the control group. In a separate study, we investigated the effects of subcutaneous administration of a lower dose of MDL (0.25 mg/kg) on ventilation in control hamsters (Schlenker EH, unpublished observations). Results from that study indicted that the lower dose decreased body temperature, but minute ventilation at baseline was maintained (frequency increased and tidal volume decreased). Moreover, there was no affect of 0.25 mg/kg MDL relative to vehicle on either CO2 production or O2 consumption, but ventilatory responses to hypoxia were decreased as was ventilation following intermittent hypoxia resulting in LTD. No effects of MDL were noted on hypercapnic responses. The dose of MDL (0.5 mg/kg) we used in the present study had tremendous effects on various physiological parameters the control group, but much fewer effects in the dystrophic and PTU-treated groups, suggesting that dysthyroid states affect serotoninergic regulation of breathing and metabolism that may not have been affected by a lower dose of MDL.
Another limitation is that we did not evaluate thyroid hormone levels in the three groups. Since we have used the animal models in previous studies (50, 51), we assumed that they would exhibit similar thyroid hormone abnormalities. Moreover, thyroid hormone measurements on PTU-treated and BIO 14.6 hamsters in a separate study indicated depressed thyroid function with low T3 levels in the dystrophic hamsters (unpublished observations).
Finally, MDL was administered systemically and also crossed the blood-brain barrier. Thus, this study does not allow us to determine where MDL acts. Using immunohistochemistry, we determined that in hamsters’ brains 5-HT2A receptors are located in raphé nucleic, nucleus of the solitary tract (NTS), hypoglossal nuclei, the lateral paragigantocellularus, nucleus, ambiguous nucleus, and ventral tegmental nuclei among others (Schlenker EH and Hansen SN, unpublished observations). These regions, such as the NTS, are important in cardiopulmonary regulation and integration of inputs from carotid body (15). Thus, MDL may have acted on 5-HT2A receptors in several regions to influence control of breathing.
Conclusions
We can draw several conclusions from the present study. Serotonin acting on 5-HT2A receptors modulated ventilation, metabolism, and body temperature in hamsters. Blocking 5-HT2A receptors in dystrophic and PTU-treated hamsters affected body temperature in the same way as in control hamsters but had dissimilar effects on ventilation relative to effects on control hamsters. With MDL treatment, following exposure to intermittent hypoxia the control group exhibited LTD as was hypothesized. However, MDL treatment did not prevent LTD in the dystrophic and PTU-treated groups as was previously hypothesized. Moreover, we did not see the same ventilatory and metabolic responses in the dystrophic and PTU-treated groups, suggesting that factors other than thyroid hormone status may contribute to these differences. Future studies will evaluate the location and quantity of 5-HT2A receptors in brain regions associated with ventilation, metabolism, and body temperature in these three groups, such as the paraventricular nucleus, NTS, and hypoglossal nuclei. In addition, determination of pAkt and brain-derived neurotrophic factor levels prior to, during, and following intermittent hypoxia may be helpful to understand their role in the development of LTD. The results of the present study raise the possibility that thyroid hormone dysfunction and muscular dystrophy may contribute to LTD in human subjects, and ventilation could be modulated differently relative to control subjects by administration of drugs that act on 5-HT2A receptors.
Perspectives and Significance
Even though the prevalence of hypothyroidism in the population is >5% and that of subclinical hypothyroidism >10%, the mechanisms responsible for frequent abnormalities in control of breathing, such as sleep apnea in these subjects are not well understood. Hypothyroidism is known to affect skeletal and cardiac muscle function and decreased vascular density, but its effects on sensing and integration signals from the carotid body to affect ventilation are unknown. This study used two animal models, the dystrophic hamster and the hypothyroid hamster and acute intermittent hypoxia protocol to investigate role of 5-HT2A receptor modulation of breathing in hypothyroidism. Our findings that hypothyroidism and blockade of 5-HT2A receptors in euthyroid hamsters results in the production of ventilatory LTD, as well as molecular models underlying ventilatory LTF presented in this paper, may help elucidate the mechanisms responsible for nervous system dysfunctions in regulation of breathing in hypothyroidism.
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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. Section 1734 solely to indicate this fact.
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