Conversion from testosterone to oestradiol is required to modulate respiratory long-term facilitation in male rats.

Sex hormones modulate plasticity in the central nervous system, including respiratory long-term facilitation (LTF), a form of serotonin-dependent respiratory plasticity induced by intermittent hypoxia. Since gonadectomy (GDX) attenuates LTF in male rats, we tested the hypotheses that: (1) testosterone replenishment restores LTF in gonadectomized male rats, and (2) that the conversion of testosterone to oestradiol (under the influence of aromatase) is required for these effects. Intact and sham operated male F344 rats were compared to gonadectomized rats implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (ADT), or 5alpha-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. Seven days postsurgery, LTF was studied in anaesthetized, neuromuscularly blocked and ventilated rats while monitoring integrated phrenic and hypoglossal (XII) motor output. LTF was elicited by three 5 min hypoxic episodes (P(a,O(2)) = 35 - 45 mmHg). Although significant phrenic and XII LTF were observed in all rat groups, GDX reduced both phrenic and XII LTF, an effect reversed by T. In contrast, LTF was not restored in T + ADT or DHT-treated gonadectomized rats. We conclude that the conversion of testosterone to oestradiol modulates phrenic and XII LTF in male F344 rats.

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

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.

Selected contribution: chronic intermittent hypoxia enhances respiratory long-term facilitation in geriatric female rats.

Age and the estrus cycle affect time-dependent respiratory responses to episodic hypoxia in female rats. Respiratory long-term facilitation (LTF) is enhanced in middle-aged vs. young female rats (72). We tested the hypothesis that phrenic and hypoglossal (XII) LTF are diminished in acyclic geriatric rats when fluctuating sex hormone levels no longer establish conditions that enhance LTF. Chronic intermittent hypoxia (CIH) enhances LTF (41); thus we further predicted that CIH would restore LTF in geriatric female rats. LTF was measured in young (3-4 mo) and geriatric (20-22 mo) female Sasco Sprague-Dawley rats and in a group of geriatric rats exposed to 1 wk of nocturnal CIH (11 vs. 21% O2 at 5-min intervals, 12 h/night). In anesthetized, paralyzed, vagotomized, and ventilated rats, time-dependent hypoxic phrenic and XII responses were assessed. The short-term hypoxic response was measured during the first of three 5-min episodes of isocapnic hypoxia (arterial Po2 35-45 Torr). LTF was assessed 15, 30, and 60 min postepisodic hypoxia. Phrenic and XII short-term hypoxic response was not different among groups, regardless of CIH treatment (P > 0.05). LTF in geriatric female rats was smaller than previously reported for middle-aged rats but comparable to that in young female rats. CIH augmented phrenic and XII LTF to levels similar to those of middle-aged female rats without CIH (P < 0.05). The magnitude of phrenic and XII LTF in all groups was inversely related to the ratio of progesterone to estradiol serum levels (P < 0.05). Thus CIH and sex hormones influence the magnitude of LTF in geriatric female rats.

Age and gender effects on serotonin-dependent plasticity in respiratory motor control.

We review recent evidence indicating that serotonin-dependent plasticity in respiratory control is influenced by age and gender. Specifically, respiratory long-term-facilitation following intermittent hypoxia decreases with age in male rats, but increases in female rats. We speculate about a possible relationship between age and gender effects on serotonin-dependent plasticity in upper airway motoneuron pools and the prevalence of obstructive sleep apnea.

Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.

The objectives of this paper are: (1) to review advances in our understanding of the mechanisms of respiratory plasticity elicited by episodic versus continuous hypoxia in short to intermediate time domains (min to h); and (2) to present new data suggesting that different patterns of hypercapnia also elicit distinct forms of respiratory plasticity. Episodic, but not continuous hypoxia elicits long-term facilitation (LTF) of respiratory motor output. Phrenic LTF is a serotonin-dependent central neural mechanism that requires: (a) activation of spinal serotonin receptors; and (b) spinal protein synthesis. Continuous and episodic hypercapnia also elicit different mechanisms of plasticity. Continuous, severe hypercapnia (25 min of approximately 10% inspired CO(2)) elicits long-term depression (LTD) of phrenic motor output (-33+/-8% at 60 min post-hypercapnia) in anesthetized rats. In contrast, 3,5 min hypercapnic episodes do not elicit LTD (9+/-17% at 60 min). We hypothesize that the response of respiratory motoneurons to serotonergic and noradrenergic modulation may contribute to pattern sensitivity to hypoxia and hypercapnia.

Selected contribution: Time-dependent hypoxic respiratory responses in female rats are influenced by age and by the estrus cycle.

Age affects time-dependent respiratory responses to episodic hypoxia in male rats, particularly long-term facilitation (LTF), a serotonin-dependent respiratory "memory" [Zabka AG, Behan M, and Mitchell GS, J Physiol (Lond) 531: 509, 2001]. Because age and gender influence serotonergic function, we tested the hypotheses that the short-term hypoxic response (STHR), posthypoxia frequency decline (PHFD) and LTF of phrenic and hypoglossal (XII) motor output change with age and stage of the estrus cycle in female rats. Young (3-4 mo) and middle-aged (13 mo) female Sprague-Dawley rats were anesthetized, paralyzed, vagotomized, and ventilated. STHR was measured during and PHFD after the first of three 5-min episodes of isocapnic hypoxia (arterial P(O)(2) 35-45 Torr). LTF was assessed 60 min postepisodic hypoxia. Phrenic and XII STHR increased with age (P < 0.05). PHFD was unaffected by age or gender. Phrenic LTF increased with age in both estrus and diestrus (P < 0.05), whereas XII LTF increased in middle-aged female rats during diestrus only. Age and gender influence time-dependent hypoxic phrenic and XII responses in a complex manner.

Histomorphometric description of allograft bone remodeling and union in a canine segmental femoral defect model: a comparison of rhBMP-2, cancellous bone graft, and absorbable collagen sponge.

The purpose of this study was to determine the effect of recombinant human bone morphogenetic protein type 2 (rhBMP-2) on the histomorphometry of femoral allograft-host bone union and allograft remodeling. A 6 cm mid-diaphyseal femoral defect was created and filled with an allograft stabilized with an interlocking nail in 21 dogs. Dogs were randomly divided into three equal groups and the allograft-host bone junctions and the mid-diaphyses of the allografts were treated with either an absorbable collagen sponge (ACS) loaded with rhBMP-2 (BMP group), an autogenous cancellous bone graft (CBG group), or ACS loaded with buffer solution (ACS group). All dogs received daily tetracycline until sacrifice at 24 weeks to label new bone formation. Histomorphometric analyses on sections of proximal and distal allograft-host bone junctions and the mid-diaphyseal portion of allografts were performed using fluorescent and regular light microscopy. Analyses of the host bone and junctions between allograft and host bone revealed significantly greater new bone formation and larger osteon radii in the BMP group compared to CBG and ACS groups and contralateral intact bone. Porosity in CBG and ACS groups was significantly higher than in the BMP group, which had similar values to intact bone. In transverse sections of allografts, the largest pore diameters were present in the CBG group. Based on all parameters measured, significantly higher bone turnover occurred in the outer cortical area of the allograft in all groups as compared to the inner cortical and mid-cortical areas. New bone formation and osteon radius/osteon width in allografts were similar for all three groups. Higher porosity and larger pore diameters in the CBG and ACS groups suggested higher bone resorption versus formation in these groups compared to the BMP group. The results of this study reveal more balanced allograft bone resorption and bone formation in the BMP group, with greater resorptive activity in the CBG and ACS groups. However, neither rhBMP-2 nor autogenous bone graft increased allograft incorporation when compared to the negative control (ACS group).

Invited review: Intermittent hypoxia and respiratory plasticity.

Intermittent hypoxia elicits long-term facilitation (LTF), a persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of the mechanisms and manifestations of this potentially important model of respiratory plasticity. LTF is elicited by intermittent but not sustained hypoxia, indicating profound pattern sensitivity in its underlying mechanism. During intermittent hypoxia, episodic spinal serotonin receptor activation initiates cell signaling events, increasing spinal protein synthesis. One associated protein is brain-derived neurotrophic factor, a neurotrophin implicated in several forms of synaptic plasticity. Our working hypothesis is that increased brain-derived neurotrophic factor enhances glutamatergic synaptic currents in phrenic motoneurons, increasing their responsiveness to bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory outputs, differs among experimental preparations, and is influenced by age, gender, and genetics. Furthermore, LTF is enhanced following chronic intermittent hypoxia, indicating a degree of metaplasticity. Although the physiological relevance of LTF remains unclear, it may reflect a general mechanism whereby intermittent serotonin receptor activation elicits respiratory plasticity, adapting system performance to the ever-changing requirements of life.

Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.

Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (integralPhr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial PO(2) = 40 +/- 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [1) control (n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia (n = 7 each)]. Ketanserin transiently decreased integralPhr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, integralPhr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.

Long term facilitation of respiratory motor output decreases with age in male rats.

Long term facilitation (LTF) is a serotonin-dependent augmentation of respiratory motor output (phrenic and hypoglossal) following episodic hypoxia. Since ageing influences respiratory control mechanisms and serotonergic function, we tested the hypothesis that LTF decreases with age in male rats. Young (3-4 month) and aged (13 month) male Sprague-Dawley rats were anaesthetized with urethane, vagotomized, paralysed and pump ventilated. Integrated phrenic and hypoglossal (XII) nerve activities were measured before (baseline), during and for 60 min after three 5 min episodes of isocapnic hypoxia (Pa,O2 35-45 mmHg) separated by 5 min of hyperoxia (Pa,O2 > 150 mmHg). In young rats, LTF was observed as an augmentation in peak integrated phrenic (n = 8) and XII (n = 7) amplitudes following episodic hypoxia (56 +/- 14 and 73 +/- 16 % (means +/- S.E.M.) at 60 min post-hypoxia, respectively; both P < 0.05). In aged rats, LTF was significantly increased compared to baseline in phrenic (25 +/- 8 % at 60 min, P < 0.05), but not in XII (4 +/- 7 %, P > 0.05) motor output. LTF was significantly greater in young than in aged rats in both motor outputs (P < 0.05). Decreased phrenic and XII LTF suggests that serotonergic modulation of respiratory motor output decreases in ageing male rats. We speculate that decreased serotonergic modulation may contribute to age-related breathing disorders.