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. 2004 Dec 21;563(Pt 2):557–568. doi: 10.1113/jphysiol.2004.077511

Ageing and gonadectomy have similar effects on hypoglossal long-term facilitation in male Fischer rats

AG Zabka 1, GS Mitchell 1, M Behan 1
PMCID: PMC1665590  PMID: 15613371

Abstract

Long-term facilitation (LTF), a form of serotonin-dependent respiratory plasticity induced by intermittent hypoxia, decreases with increasing age or following gonadectomy in male Sprague-Dawley (SD) rats. Ageing is accompanied by decreasing levels of testosterone, which in turn influences serotonergic function. In addition, LTF in young male rats differs among strains. Thus, we tested the hypothesis that LTF is similar in middle-aged and gonadectomized young male rats of an inbred rat strain commonly used in studies on ageing (F344) by comparison with SD rats. We further tested whether the magnitude of LTF correlates with circulating serum levels of testosterone and/or progesterone. Young and middle-aged intact and young gonadectomized (GDX) male Fischer 344 rats were anaesthetized, neuromuscularly blocked and ventilated. Integrated phrenic and hypoglossal (XII) nerve activities were measured before, during and 60 min following three 5-min episodes of isocapnic hypoxia. LTF was observed in phrenic motor output in young and middle-aged intact and young GDX rats. In contrast, XII LTF was observed only in young intact rats. In middle-aged and young GDX rats, XII LTF was significantly lower than in young intact rats (P < 0.05). Furthermore, XII LTF was positively correlated with the testosterone/progesterone ratio. These data show that serotonin-dependent plasticity in upper airway respiratory output is similar in F344 and SD rat strains. Furthermore, LTF is similarly impaired in middle-aged and gonadectomized male rats, suggesting that gonadal hormones play an important role in modulating the capacity for neuroplasticity in upper airway motor control.

Ageing alters responses to respiratory challenges. For example, responses to hypoxia or hypercapnia are diminished in aged rats (Fukuda 1991, 1992). Ageing also alters the production of gonadal hormones. For example, testosterone levels decrease progressively with age in males (humans and other mammals) (Goudsmith et al. 1990; Ferrini & Barrett-Connor, 1998). In a previous study, we demonstrated that ageing decreases long-term facilitation (LTF) of respiratory motor output following episodic hypoxia in male Sprague-Dawley (SD) rats (Zabka et al. 2001). In a different study, a similar effect was seen following gonadectomy in young male SD rats (Behan et al. 2003). In this study, we investigated whether decreased testosterone due to ageing or gonadectomy have a comparable effect on LTF in an inbred rat strain, Fischer 344, as these rats are commonly used to study the effects of ageing.

LTF is a long-lasting augmentation of respiratory motor output following intermittent hypoxia that is observed in several awake and anaesthetized mammalian species (Mitchell et al. 2001). LTF can be elicited by episodic hypoxia or electrical stimulation of carotid chemoafferent neurones, which indicates a central neural mechanism (Millhorn et al. 1980; Mitchell et al. 2001; Feldman et al. 2003). In anaesthetized rats, LTF of integrated phrenic and hypoglossal (XII) motor output is commonly observed as an augmentation of nerve burst amplitude, with a smaller, more variable increase in burst frequency (Bach & Mitchell, 1996; Powell et al. 1998; Fuller et al. 2000). LTF requires serotonin receptor activation and BDNF protein synthesis within the respective motor nuclei following the hypoxic episodes (Baker-Herman & Mitchell, 2002; Baker-Herman et al. 2004). Since spinal serotonin receptor activation during the hypoxic episodes initiates LTF, serotonin plays a critical role in orchestrating the events leading to plasticity (Fuller et al. 2001; Baker-Herman et al. 2004).

Serotonergic function is influenced by age and by sex hormone levels. For example, serotonin immunoreactivity decreases in the XII nucleus of middle-aged versus young male Fischer rats (Behan & Brownfield, 1999). Furthermore, 5-HT decreases with age in the ventral horns of cervical spinal segments associated with the phrenic motor nucleus (Ko et al. 1997). Oestrogen increases 5-HT levels through multiple mechanisms (Poirier et al. 1985; Lopez-Jaramillo & Teran, 1999; Bethea et al. 2000), and gonadectomy in female rats decreases 5-HT immunoreactivity in the XII motor nucleus (M. Behan, unpublished observations). Testosterone also affects the serotonergic system, and gonadectomy in male rats reduces 5-HT in the caudal raphe nucleus (Long et al. 1983). Thus, age-related changes in the serotonergic system and its influence on respiratory motor control could occur indirectly via changes in levels of gonadal hormones.

Since the magnitude of LTF varies among rat strains (Fuller et al. 2000, 2001; Bavis et al. 2003), critical factors that influence time-dependent hypoxic responses differ between rat strains, and these factors may be altered in unique strain-dependent ways by age and sex hormones. After discontinuation of the particular colony (K62) of SD rats, we focused our interest on F344 rats that are readily available at any age to study effects of ageing. In this study, we tested the hypothesis that hypoxic ventilatory responses are similar in young, middle-aged and gonadectomized male F344 compared to SD rats before investigating underlying mechanisms of sex hormone influences on respiratory control. Furthermore, we tested the hypothesis that circulating sex hormones levels correlate with the expression of LTF in male F344 rats as in female SD rats (Zabka et al. 2003).

The effects of age and sex hormones on hypoxic responses have seldom been investigated, particularly their impact on respiratory plasticity following intermittent hypoxia (Behan et al. 2002, 2003). Such an understanding is of fundamental interest since several breathing disorders, including obstructive sleep apnoea, exhibit age- and sex-related patterns in their prevalence (Bixler et al. 2001; Ware et al. 2000).

Methods

Experimental groups

Experiments were performed on male Fischer 344 rats from the NIH-NIA Aged Rodent Colony. Fischer 344 rats are commonly used to study ageing. They are bred by the National Institute of Ageing and are made available to investigators in a range of ages to facilitate studies on the biology of ageing. Three groups of male rats were used for this study: intact young adult rats (Young, 3–4 months, n = 5, 311 ± 20 g), young adult gonadectomized rats (GDX, 3–4 months, n = 6, 256 ± 9 g), and middle-aged intact rats (Middle-Aged, 19 months, n = 5, 422 ± 12 g). All experimental procedures were approved by the University of Wisconsin-Madison Animal Care and Use Committee.

Gonadectomy

Rats were briefly anaesthetized with isoflurane in an induction chamber and maintained (2.0–2.5% isoflurane in 30% O2) via a nose cone. To prevent post-surgical pain, rats received 0.05 mg kg−1 buprenorphine i.m. prior to surgery. Rats were positioned in dorsal recumbency on a heated pad. The scrotum was clipped and washed with an antiseptic detergent. Skin incisions of 1 cm were made over the testicles followed by blunt spreading of the muscle layer. Testicles were removed by separating them distally from a ligature around the ductus deferens and accompanying blood vessels with a scalpel. The muscle layer and the skin incision were closed with reabsorbale suture and staples, respectively. After initiating anaesthesia for the acute neurophysiological experimental protocol (see experimental preparation) 7 days post-gonadectomy, blood was collected to assess levels of testosterone, oestrogen and progesterone. Hormone levels were compared to levels in intact animals that were used for the neurophysiological experiments.

Experimental preparation

Anaesthesia was initiated with isoflurane in an induction chamber and maintained (3.0–3.5% isoflurane in 50% O2, balance N2) first through a nose cone, and continued through a tracheal cannula placed to allow artificial ventilation (Small Animal Ventilator, Model 683, Harvard Apparatus Inc., Holliston, MA, USA). A hyperoxic gas mixture was used to stabilize the anaesthetic preparation for a longer time frame than possible under normoxia. Isoflurane anaesthesia was slowly converted to urethane (1.6 g kg−1) through an intravenous catheter placed in a femoral vein. Supplemental urethane was administered as necessary to prevent blood pressure responses to toe pinch. Blood pressure was monitored through a femoral arterial catheter, and discrete blood samples (0.2 ml in a 0.5 ml heparinized glass syringe) were drawn to determine arterial blood gases (Pa,O2 and Pa,CO2), pH and base excess (ABL 500; Radiometer, Copenhagen, Denmark). Arterial blood values were corrected to the body temperature. Body temperature was maintained between 37 and 38°C using a heated table.

To prevent spontaneous breathing efforts and entrainment with the ventilator, all animals were bilaterally vagotomized and neuromuscularly blocked (pancuronium promide, 2.5 mg kg−1i.v.). End-tidal CO2 was measured with a flow-through capnograph (Capnogard, Novametrix; Wallingford, CT, USA), with sufficient response time to measure end-expiratory gases in rats.

The left phrenic and XII nerves were isolated via a dorsal approach, cut distally, desheathed, submerged in mineral oil and placed on bipolar silver wire electrodes. Nerve activities were amplified (× 10 000), band-pass filtered (100 Hz to 10 kHz) (Model 1700, A-M Systems, Inc.; Carlsborg, WA, USA) and integrated (time constant = 50 ms, Model MA-821RSP, CWE Inc., Ardmore, PA, USA). Integrated nerve signals were digitized and processed with computer software (WINDAQ software, DATAQ Instruments, Akron, OH, USA). To terminate an experiment, rats were killed by an overdose of urethane i.v.

Experimental protocol

Nerve signals were allowed to stabilize for approximately 60–90 min following surgery under hyperoxic (Pa,O2 > 150 mmHg, Table 1) and normocapnic conditions. In previous studies, we noted that moderate hyperoxia prolongs preparation viability relative to normoxic conditions, but does not alter the expression of respiratory LTF (Bavis & Mitchell, 2003). The CO2 apnoeic threshold was then determined by increasing the ventilation rate to lower Pa,CO2 until phrenic nerve activity ceased, and then by decreasing ventilation until rhythmic phrenic nerve activity resumed. The latter value was assigned as the CO2 apnoeic threshold. Baseline nerve activities were established by increasing Pa,CO2 2–3 mmHg above this CO2 apnoeic threshold. Baseline blood gas values were assessed before starting the protocol. All subsequent blood samples were compared to this initial baseline value. Strict isocapnic conditions (± 1 mmHg from baseline Pa,CO2) were maintained throughout an experiment by monitoring end-tidal CO2 and making adjustments in ventilation rate and/or inspired CO2 as necessary. To prevent alveolar atelectasis, lungs were hyperinflated approximately every 60 min.

Table 1.

Partial pressure of arterial O2 (mmHg) in Young (n = 5), Middle-Aged (n = 5), and GDX (n = 6) rats at different time points

Pa,O2 Baseline 1. Hypoxia 15 min 30 min 60 min
Young 222 ± 20 37 ± 2 197 ± 18 230 ± 15 212 ± 20
Middle-aged 224 ± 15 41 ± 2 182 ± 15 223 ± 14 230 ± 8
GDX 233 ± 10 38 ± 2 171 ± 14 226 ± 19 211 ± 17
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Data are means ± s.e.m. Strict isocapnic conditions were maintained.

A typical tracing of phrenic motor output of a young intact male rat is shown in Fig. 1. After establishing a stable baseline, the protocol started with three episodes of hypoxia of 5 min duration (FI,O2 = 0.11–0.12, target Pa,O2 35–45 mmHg), separated by 5 min intervals, and followed by 60 min of isocapnic hyperoxic baseline conditions. A protocol ended with 5 min of hypercapnia (PET,CO2 = 80–90 mmHg) to assess maximal nerve activity. Arterial blood samples were drawn at 15, 30 and 60 min after the final hypoxic episode to confirm isocapnic conditions. Rats were excluded from analysis if Pa,CO2 deviated by more than 1 mmHg from baseline. Therefore, changes in Pa,CO2 had minimal impact on the results of this study. Experiments were also excluded if arterial blood pressure dropped by more than 30 mmHg from baseline at the end of a protocol.

Figure 1. Neurograms of representative LTF protocols showing integrated XII motor output in Young, Middle-Aged and gonadectomized (GDX) rats.

Figure 1

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A, a typical example from a young male rat exhibiting approximately 70% LTF at 60 min post-episodic hypoxia. B, from a middle-aged, and C, from a gonadectomized male rat, both showing no LTF. Arrows indicate time points at which data were analysed and blood samples taken: baseline (BL), last minute of first hypoxic episode (H1), 15, 30 and 60 min post-episodic hypoxia, and hypercapnic challenge (HC) to assess maximal nerve activity. Baseline conditions were established under hyperoxia (50% O2) to achieve long lasting stable anaesthesia conditions.

Sex hormone levels

Arterial blood samples (1 ml) were taken as soon as the arterial catheter was placed. Subsequently, blood samples were centrifuged to collect serum. Serum was immediately frozen at −70°C. After collection of all serum samples, testosterone, oestradiol and progesterone levels were analysed using radio immunoassay (RIA) (Testosterone Coat-A-Count, Oestradiol Coat-A-Count, Progesterone Coat-A-Count; Diagnostic Products, Los Angeles, CA, USA). The RIA method measures free (versus total) sex hormone concentrations. Prior to analysing the samples, the assays were validated with pooled serum from 10 rats. Since serum levels of sex hormones differ among species and rat strains, it is desirable to generate a standard curve for that species and strain. This is particularly important when hormone levels are low.

Data analysis

Phrenic and XII nerve activities were recorded throughout the protocol. Peak integrated amplitude (∫Phr and ∫XII), burst frequency (bursts min−1), and mean arterial blood pressure (MAP) were measured at the following time points (Fig. 1): baseline, last minute of first hypoxic episode (short-term hypoxic response), 15, 30 and 60 min after the final hypoxic episode, and the last minute of the hypercapnic response (max. CO2 response). Nerve activity was averaged over approximately 60 s in each condition. Changes in amplitude from baseline were normalized as a percentage of baseline nerve activity (% baseline) and as a percentage of the hypercapnic response (% maximum). All conclusions were the same, regardless of the normalization used. Thus, only percentage baseline data are presented in this paper. Changes in burst frequency were expressed as a difference from baseline in bursts min−1. Phrenic and XII minute activity was the product of integrated peak amplitude and burst frequency.

Depending on the variable, either a one-way or a two-way ANOVA with a repeated measures design (SigmaStat version 2.0, Jandel Corporation, San Rafael, CA, USA) was performed followed by a post hoc least significant difference test for individual comparisons. Differences were considered significant if P < 0.05. All data reported are means ± s.e.m. Serum levels of testosterone, oestradiol and progesterone in individual rats were related to the magnitude of phrenic and XII LTF via multiple and/or simple linear regressions. A variable was considered to contribute significantly to the model if P < 0.05.

Results

Experimental animals

Mean weights of rats in the three groups were significantly different (P < 0.01). Despite the same age and similar weight before the start of experiments, gonadectomized rats (256 ± 9 g) were lighter than intact rats (311 ± 20 g) 7 days following gonadectomy. Middle-aged rats (422 ± 12 g) were heavier than both groups of young rats.

Apnoeic threshold, CO2 regulation and baseline conditions

The CO2 apnoeic threshold was similar in all three rat groups (P > 0.05). Baseline conditions were standardized through individual determinations of the CO2 apnoeic threshold, and establishing baseline conditions 2 mmHg above this level. Throughout the protocols, strict isocapnic conditions (Pa,CO2± 1 mmHg to baseline values) were maintained at this baseline value. Therefore, changes in Pa,CO2 were prevented from influencing phrenic or XII nerve activity. Resting burst frequency did not differ among groups (Young = 44 ± 1; GDX = 44 ± 3; Middle-Aged = 54 ± 5 bursts min−1; P = 0.084). The ratio of baseline/maximal CO2 response was not different among Young, Middle-Aged and GDX rats for phrenic and XII nerve activity indicating a similar dynamic range and baseline ventilatory drive in all rats (Phrenic: Young = 0.49 ± 0.03; GDX = 0.57 ± 0.06; Middle-Aged = 0.38 ± 0.07; XII: Young = 0.28 ± 0.02; GDX = 0.34 ± 0.06; Middle-Aged = 0.36 ± 0.06; P > 0.05).

Short-term hypoxic responses (STHR)

Pa,O2 during hypoxic episodes did not differ among rat groups (Young = 37 ± 2, GDX = 38 ± 2, Middle-Aged = 41 ± 2 mmHg; P > 0.05). Phrenic STHR, expressed as a percentage change from baseline (Δ percentage), was significantly greater in Middle-Aged rats than Young or GDX rats (P < 0.05; Fig. 2A). In contrast, XII STHR was not different among rat groups (P > 0.05).

Figure 2. Short-term hypoxic response (STHR) of phrenic and hypoglossal motor output in Young, Middle-Aged and gonadectomized (GDX) rats.

Figure 2

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STHR was measured as an amplitude increase in integrated phrenic and hypoglossal nerve activity from baseline (Δ∫Phr and Δ∫XII; % baseline). A, phrenic short-term hypoxic responses was significantly greater in Middle-Aged versus Young and GDX rats (P = 0.01). XII STHR was not different among the three groups (P > 0.05). B, the change in frequency of the STHR during hypoxia was not different among the three groups (P > 0.05). Values are means ± s.e.m.*Middle-Aged was significantly different from Young and GDX.

The respiratory burst frequency, measured during the last 2 min of the first hypoxic episode, was not significantly different among groups (Fig. 2B; P > 0.05).

Phrenic long-term facilitation (LTF)

In all three groups, ∫Phr increased progressively following episodic hypoxia, indicating the development of phrenic LTF (P < 0.001; Fig. 3A). Phrenic burst amplitude expressed as a percentage change from baseline (Δ percentage BL) was significantly elevated above baseline in middle-aged and gonadectomized rats at 30 min, and in all three groups at 60 min postepisodic hypoxia (P < 0.05).

Figure 3. Phrenic and hypoglossal long-term facilitation (LTF) in Young, Middle-Aged and GDX rats.

Figure 3

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LTF was measured as an amplitude increase in integrated phrenic (A) and XII (B) nerve activity from baseline (Δ∫Phr and Δ∫XII,% baseline) and burst frequency (C; Δf, breaths min−1) at 15, 30 and 60 min after the final hypoxic episode. Phrenic motor output was significantly elevated above baseline at 30 min in Young and GDX, and at 60 min in all three groups of rats indicating LTF (P < 0.05). Phrenic LTF was not different among the three groups at any time measured (P > 0.05). XII motor output was only significantly elevated above baseline at 30 and 60 min following episodes of hypoxia in Young rats indicating LTF (P < 0.05). There was no XII LTF expressed in. Middle-Aged or GDX rats at any time post-episodic hypoxia. At 60 min, XII motor output was significantly greater in Young versus Middle-Aged and GDX rats (P < 0.01). All three groups developed frequency LTF expressed as a significant increase of frequency versus baseline following episodes of hypoxia. There were no differences in frequency LTF among groups (P > 0.05). Values are means ± s.e.m.#Significantly different from baseline. *Significantly different among groups.

XII long-term facilitation

In a two-way ANOVA, there was a significant time–treatment interaction in XII nerve activity (P = 0.029). In young intact rats, ∫XII increased progressively following episodic hypoxia, indicating XII LTF; ∫XII was significantly elevated above baseline at 30 and 60 min post-hypoxia (P < 0.05). In contrast, LTF (i.e. significant increase in burst amplitude from baseline) was not observed in gonadectomized and middle-aged rats at any time post-episodic hypoxia (P > 0.05; Fig. 3B). At 60 min post-hypoxia, XII LTF was significantly greater in young intact versus gonadectomized or middle-aged rats (both P < 0.001).

Frequency long-term facilitation

Following hypoxic episodes, all groups of rats showed small but significant increases in burst frequency versus baseline (i.e. frequency LTF; Young and Middle-Aged at 30 min, all groups at 60 min; P < 0.05; Fig. 3C). There were no differences among groups at any time measured.

Long-term facilitation of phrenic and XII minute activity

In all three groups, phrenic minute activity increased progressively following episodic hypoxia, indicating the development of LTF (P < 0.05). At 60 min post-hypoxia, phrenic minute activity was similar among the three groups (P > 0.05; Fig. 4). LTF of XII minute activity was detected only in young intact rats (P < 0.05). XII minute activity was significantly greater in young intact versus middle-aged or gonadectomized rats (P < 0.05: Fig. 4).

Figure 4. LTF of phrenic and hypoglossal minute neural activity in Young, Middle-Aged and GDX rats measured at 60 min following intermittent hypoxia.

Figure 4

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Changes in phrenic and XII minute activity were measured as a percentage increase versus prehypoxic baseline minute activity. At 60 min post-intermittent hypoxia, phrenic minute activity was not different among the three groups (P > 0.05), but XII minute activity was significantly greater in Young versus Middle-Aged and GDX rats (P < 0.05). Values are means ± s.e.m.*Young significantly greater than Middle-Aged and GDX.

Sex hormone levels

Seven days post-gonadectomy, serum testosterone levels (ng ml−1) were nearly undetectable, and were significantly lower than in intact young and middle-aged rats (P < 0.05; Fig. 5A). Middle-aged rats had significantly lower serum testosterone levels than young intact rats (P < 0.05; Fig. 5A). Oestradiol levels (pg ml−1) were significantly higher in middle-aged versus young gonadectomized rats (P < 0.05; Fig. 4B), but were not different from young intact rats. Progesterone levels (ng ml−1) were unaffected by age or gonadectomy (P > 0.05; Fig. 5C).

Figure 5. Serum levels of testosterone, oestradiol and progesterone.

Figure 5

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A, testosterone levels (ng ml−1) were nearly undetectable in gonadectomized (GDX) rats and significantly reduced in Middle-Aged versus Young rats (P < 0.05). B, oestradiol levels (pg ml−1) were significantly greater in Middle-Aged versus GDX rats (P < 0.05). C, progesterone levels (ng ml−1) did not differ among groups (P > 0.05). Values are means ± s.e.m.*Significantly different among groups.

There was a significant correlation between XII LTF and testosterone alone (Fig. 6A), and between XII LTF and the ratios of testosterone to progesterone (T/P) (Fig. 6B) and testosterone to oestradiol (T/E) (Fig. 6C; all P < 0.05). The linear models expressing these relationships were:

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Figure 6. The magnitude of XII LTF was correlated with serum levels of testosterone, progesterone and oestradiol.

Figure 6

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XII LTF measured at 60 min post-episodic hypoxia was positively correlated to testosterone (A), to the ratio of testosterone and progesterone (B), and to ratio of testosterone to oestradiol in all three experimental groups (C) (P < 0.05).

There was no significant correlation between phrenic LTF and any sex hormone or sex hormone ratio measured.

Mean arterial blood pressure (MAP)

MAP was similar in all groups of rats at any time before, during and following hypoxic episodes (P > 0.05, Fig. 7). During hypoxic episodes, MAP decreased in all three groups, as is typical in hypoxic anaesthetized rats.

Figure 7. Mean arterial blood pressure (MAP) did not differ among groups.

Figure 7

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MAP was measured under baseline conditions (BL), during the first of three hypoxic episodes (H1), and 15, 30 and 60 min after the final hypoxic episode. MAP dropped during hypoxia in all groups as is common in anaesthetized rats. Values are means ± s.e.m.

Discussion

Age and gonadectomy both diminish XII long-term facilitation similarly in male Fischer 344 rats. On the other hand, phrenic LTF was not affected by either factor in this rat strain. These results contrast with previous findings in middle-aged and gonadectomized male Sprague-Dawley rats which showed diminished phrenic, as well as XII, LTF (Zabka et al. 2001; Behan et al. 2003). Thus, although sex hormones may exert a more powerful influence on XII plasticity, this difference may depend on underlying genetics as reflected in rat strain.

Experimental animals

At the time the experiments were performed, the mean weight differed significantly among rat groups. However, by directly recording phrenic and XII nerve activity to assess neural drive, intrinsic factors such as body mass should not have influenced our results. On the other hand, middle-aged rats may have more fat tissue, which may produce small amounts of oestradiol (Nelson & Bulun, 2001). Therefore, fat tissue-derived oestradiol in middle-aged rats may have minimized age effects on LTF (see also Sex hormone levels, below).

In recent experiments on the same rat stain, sham- operated animals exhibited no significant differences from intact rats in any variable measured (Zabka et al. 2004), thus providing assurance that surgical stress played minimal, if any, role in the experimental outcome.

Short-term hypoxic response (STHR)

Ventilatory responses to hypoxia are diminished with age (Peterson et al. 1981; Fukuda, 1992; Serebrovskaya et al. 2000; Pokorski & Marczak, 2003). However, as phrenic motor output was measured in our studies, the influence of age-related changes in lung mechanics or gas exchange was eliminated. The enhanced phrenic STHR in middle-aged versus young intact or gonadectomized Fischer 344 rats differed from earlier observations in middle-aged, young and gonadectomized male Sprague-Dawley rats that phrenic STHR was similar among groups (Zabka et al. 2001; Behan et al. 2003). Even in rats of similar age, hypoxic responses vary among inbred rat strains (Strohl et al. 1997; Hodges et al. 2002; Bavis et al. 2003). Our data indicate that age effects on the phrenic STHR also vary among rat strains, suggesting that age-dependent changes in hypoxic responses may be influenced by genetic background.

Previous studies have shown that the hypoxic ventilatory responses can be affected by gonadal hormones. Castrated male and female cats had reduced hypoxic carotid sinus nerve and ventilatory response that could be restored by administration of oestradiol and progesterone (Hannhart et al. 1990; Tatsumi et al. 1997). In the present study, the pattern of serum levels of oestradiol and progesterone was similar to the magnitude of phrenic STHR in all rat groups, suggesting that oestrogen and progesterone might selectively influence phrenic STHR. In contrast, the XII STHR seemed unaffected by age or gonadal hormones. The basis for this apparent difference remains unknown, although it points out that XII and phrenic motor output may be differentially affected by gonadal hormones.

The STHR was measured during the last 2 min of the first 5-min hypoxic episode. At this time point, the increase in amplitude is stable. However, the severity of the frequency roll-off varies considerably in individual rats, independent of age or hormonal status. Thus, the variability in the frequency response to hypoxia is large.

Taken together, age and gonadectomy appear to affect the phrenic hypoxic response differentially in male SD versus F344 rats. In contrast, the XII hypoxic response is similar in these strains.

Long-term facilitation (LTF)

LTF is commonly evoked in Sprague-Dawley rats from Sasco/Charles River (Bach & Mitchell, 1996; Fuller et al. 2000, 2001). In male rats of this particular substrain, LTF decreases substantially with age (Zabka et al. 2001) and after gonadectomy (Behan et al. 2003). However, neither age nor gonadectomy affected LTF of neither phrenic amplitude nor minute activity in Fischer 344 rats, suggesting that the capacity of phrenic motor output to develop LTF is more robust in this strain. On the other hand, LTF of XII amplitude and minute activity decreased in response to advancing age and/or diminished sex hormone levels in this strain. Genetic factors influence the magnitude of phrenic and XII LTF in young adult male rats (Fuller et al. 2001; Bavis et al. 2003). To our knowledge, this is the first information available indicating strain differences in the effects of age and/or sex hormone levels on phrenic and XII LTF.

Serotonergic (5-HT) immunoreactivity decreases with age in the XII motor nucleus of Fischer 344 rats (Behan & Brownfield, 1999), and 5-HT concentrations in this strain decrease in the cervical spinal segments associated with the phrenic nucleus (Ko et al. 1997). The number of serotonergic neurones in the raphe nuclei is maintained in aged male Wistar rats (Van Luijtelaar et al. 1992). If this is a common feature across rat strains, age-related processes are more likely to be at the level of motor nuclei or other raphe targets. Since 5-HT receptor activation is necessary to initiate LTF in Sprague-Dawley rats (Bach & Mitchell, 1996; Fuller et al. 2001), we speculate that decreased serotonergic innervation due to ageing and/or loss of gonadal hormones in the phrenic and XII motor nuclei is at least partially responsible for decreased serotonin-dependent LTF following intermittent hypoxia. Consistent with these hypotheses is the finding that gonadectomy decreases 5HT immunoreactivity in the XII nucleus of young male Sprague-Dawley rats (M. Behan & C. F. Thomas, unpublished observations).

Phrenic motoneurones appear to be less affected by gonadal hormones than XII motoneurones, depending on the rat strain. Serotonergic modulation within respiratory motor nuclei in aged or testosterone-depleted male rats could occur at different sites by: (1) morphological or neurochemical alterations at the synapse, such as decreased serotonin terminal density, reduced receptor density, or changes in the synthesis, release, reuptake and/or degradation of 5-HT; (2) alterations in downstream signalling that affect translation and/or transcription of proteins such as brain derived neurotrophic factor (BDNF) that are necessary for LTF in Sprague-Dawley and F344 rats (Baker-Herman et al. 2004); or (3) presynaptic changes in excitatory or inhibitory effector molecules such as glutamate or GABA. The site of testosterone effects on LTF is not known at this time.

Since most actions of testosterone on the serotonergic system are thought to be mediated by oestradiol, produced by the action of aromatase on testosterone (Celotti et al. 1991; Fink et al. 1998), testosterone per se may not be responsible for alterations in the magnitude of LTF. Possible sites where sex hormones could exert their effects include motor neurones and/or interneurones in the respective respiratory motor nuclei and/or serotonergic neurones in the caudal raphe nuclei that project to the XII and/or phrenic motor nuclei (Manaker et al. 1992; Manaker & Tischler, 1993).

Sex hormone levels

Testosterone levels decreased significantly with age (56%) in Fischer 344 rats, as has been reported previously for other rat strains as well as humans (Smith et al. 1992; Vom Saal et al. 1994; Luboshitzky et al. 2003; Tenover, 2003). Since testosterone production in gonadectomized rats was nearly abolished, it was not surprising that oestradiol levels were low, presumably due to a lack of testosterone available to be aromatized. However, it was surprising to find higher oestradiol levels in middle-aged rats than in young rats. Aromatase activity may be higher in the middle-aged rats. Alternatively, other tissues such as adipose tissue could have contributed to additional oestradiol production in middle-aged rats (Nelson & Bulun, 2001).

Serum testosterone (T) alone but also the balance between T and progesterone (P) or oestradiol (E) was significantly correlated with the magnitude of XII LTF. In other words, XII LTF was correlated to a specific ratio of T and P or E. Thus, not only one sex hormone alone but also a certain sex hormone ratio appears to optimize developing and/or enhancing XII LTF. Previous studies in our laboratory have shown that the P : E ratio in female rats is positively correlated with LTF in both XII and phrenic motor output (Zabka et al. 2003).

Earlier studies on ovariectomized cats demonstrated that the respiratory response to progesterone is mediated by an oestrogen-dependent mechanism (Bayliss et al. 1990), suggesting an optimizing balance between these two hormones.

The role of testosterone in the control of breathing is not well understood. Most actions of testosterone in the brain are exerted indirectly by its conversion to oestradiol (Celotti et al. 1991; Fink et al. 1998). Thus, the T to P ratio in males may at least partially reflect the E to P ratio and its effects on LTF seen in female rats. Moreover, XII LTF seems to be more reactive to certain hormone ratios than phrenic LTF in both male and female rats.

Measurements of serum sex hormone levels reflect circulating levels, but not necessarily the level in respiratory-related CNS areas such as the caudal raphe, and the phrenic or XII nuclei. Unpublished data from our laboratory suggest a widespread occurrence of androgen and oestrogen receptors in identified motoneurones within these motor nuclei. Further investigations are warranted to determine whether sex hormone levels and/or expression of their receptors in relevant regions of the brain and spinal cord underlie the effects observed in this study.

Effects of sex hormones on respiratory LTF are possibly mediated via morphological and/or neurochemical changes in or near respiratory motoneurones. Both, oestrogen and testosterone increase synaptic density and the number of dendrites and dendritic spines in hippocampal CA1 pyramidal cells and hippocampal cell cultures (Woolley & McEwen, 1994; Brinton, 2001; Leranth et al. 2004). These actions are mediated by NMDA receptor activation and GABAergic disinhibition, both of which play an important role in neuronal plasticity (McEwen & Alves, 1999). Since phrenic and XII motoneurones express androgen and oestrogen receptors, sex hormones could directly affect the functional morphology of respiratory motoneurones. Sex hormones, specifically progesterone, could exert their effects at sites that are either directly or indirectly synaptically connected to respiratory motoneurones, premotor neurones, or their neuromodulatory inputs, such as the hypothalamus (Bayliss & Millhorn, 1992).

Sex hormones could also affect brain derived neurotrophic factor (BDNF), a key molecule in eliciting LTF (Baker-Herman et al. 2004). Spinal application of BDNF (without hypoxia) is sufficient to elicit phrenic LTF and new BDNF synthesis is necessary for phrenic LTF (Baker-Herman et al. 2004). Levels of BDNF are affected by sex hormones. For example, oestrogen and progesterone treatment in ovariectomized rats increases BDNF in several brain regions (Gibbs, 1999), hippocampal BDNF fluctuates with the oestrus cycle in female rats (high oestrogen reflects high BDNF; Scharfman et al. 2003) and spinal BDNF is reduced after gonadectomy in a chronic pain model (Zhao et al. 2004). The actions of oestrogen on BDNF are thought to be mediated by an oestrogen response element encoding sequence in the BDNF gene (Sohrabji et al. 1995). Hence, oestrogen per se, or testosterone after its conversion by aromatase might increase BDNF in respiratory motoneurones and therefore enable LTF to occur.

Physiological significance

Although the physiological significance of respiratory LTF remains unknown (Mitchell et al. 2001; Feldman et al. 2003), LTF provides an excellent model to investigate respiratory neuroplasticity, and how ageing and alteration in sex hormones affect such plasticity. It has been speculated that respiratory LTF stabilizes breathing under physiological and/or pathological conditions, minimizing the impact of unintended respiratory depression (e.g. during hypoxia) or preventing repetitive apnoeas. Although our data do not address such a role directly, the age- and sex-related pattern of change in respiratory LTF has substantial similarity with the demographics of the prevalence of important breathing disorders such as obstructive sleep apnoea (OSA), suggesting a possible link between the two.

OSA is caused by narrowing of the upper airway due to relaxation of muscle tone and prolapse of the tongue during sleep. Whereas the tongue muscles receive their major innervation by the XII nerve, diaphragmatic function (and phrenic nerve activity) is less affected in OSA patients. OSA occurs mainly in middle-aged men and post-menopausal women when not on hormone replacement therapy (Hla et al. 1994; Redline et al. 1994; Ware et al. 2000; Bixler et al. 2001). Ageing is correlated with a decrease in sex hormones: a progressive decline of testosterone in men, and a more abrupt decrease of ovarian hormones in women when they reach menopause. Thus, sex hormones seem to have a role in protecting against OSA. Middle-aged and testosterone-depleted F344 rats both revealed impaired responses to repetitive hypoxia versus young intact rats, most particularly in the XII nerve. Thus, testosterone, or its conversion to oestradiol within the brain, seems to play an important role in modulating the capacity for plasticity in upper airway motor output. On this basis, we speculate that a diminished capacity for LTF – especially XII LTF – may contribute to breathing disorders such as OSA. Since XII LTF has the potential to stabilize the upper airway and maintain upper airway patency, its age-dependent loss would thus destabilize airway patency. The relationship between LTF in upper airway respiratory muscles (Babcock & Badr, 1998; Aboubakr et al. 2001) and OSA in sleeping humans requires further investigation.

Acknowledgments

This study was supported by NIH grants: AG18760 and HL 65383. The authors thank J. Armstrong, Department of Comparative Biosciences, UW, for his excellent work on the RIA analyses and M. K. Clayton, Departments of Statistics and Plant Pathology, UW, for his expert evaluation of the statistical analyses.

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