Post-hypoxic recovery of respiratory rhythm generation is gender dependent

Abstract

The preBötzinger complex (preBötC) is a critical neuronal network for the generation of breathing. Lesioning the preBötC abolishes respiration, while when isolated in vitro, the preBötC continues to generate respiratory rhythmic activity. Although several factors influence rhythmogenesis from this network, little is known about how gender may affect preBötC function. This study examines the influence of gender on respiratory activity and in vitro rhythmogenesis from the preBötC. Recordings of respiratory activity from neonatal mice (P10-13) show that sustained post-hypoxic depression occurs with greater frequency in males compared to females. Moreover, extracellular population recordings from the preBötC in neonatal brainstem slices (P10-13) reveal that the time to the first inspiratory burst following reoxygenation (TTFB) is significantly delayed in male rhythmogenesis when compared to the female rhythms. Altering activity of ATP sensitive potassium channels (KATP) with either the agonist, diazoxide, or the antagonist, tolbutamide, eliminates differences in TTFB. By contrast, glucose supplementation improves post-hypoxic recovery of female but not male rhythmogenesis. We conclude that post-hypoxic recovery of respiration is gender dependent, which is, in part, centrally manifested at the level of the preBötC. Moreover, these findings provide potential insight into the basis of increased male vulnerability in a variety of conditions such as Sudden Infant Death Syndrome (SIDS).

Figure 1. Hypoxia and rexoygenation cause stereotypical changes in rhythmic population activity.

Analysis of the in vitro preBötC rhythm was segmented according to neuronal population activity and the state of oxygenation prior to, during, and following hypoxia. (1) steady-state rhythmogenesis in carbogen prior to hypoxia; (2) augmentation during initial hypoxia (0 to 200 sec) (3) steady-state rhythmogenesis during hypoxia (i.e. last 400 sec of hypoxia) (4) TTFB upon reoxygenation; (5) post-hypoxic rhythmogenesis. Inset: The transverse preBötC slice as observed under brightfield low magnification (left). A schematic diagram of the preBötC slice and anatomical landmarks (right). The diagram labels the hypoglossal nucleus (XII), the nucleus ambiguus (NA),and the ventral respiratory column (VRC) containing the preBötC.

Figure 2. In vivo Gender differences exist in the occurrence of post-hypoxic apnea following exposure to severe hypoxia.

Representative traces of integrated electromyogram recordings from both a male (top) and female (bottom) subject. All males (n = 8) exhibited a post-hypoxic apnea following reoxygenation from severe hypoxia (95% N₂, 5% CO₂), whereas only 28% of females (n = 3 of 11) exhibited a post-hypoxic apnea.

Figure 3. Gender influences rhythm generation during the transition from a well-oxygenated state to hypoxia.

(A) Male preBötC rhythms (top) tend to fail more frequently compared to female rhythm (bottom). Asterisks denote detected integrated population bursts and scale bar repesents 20 sec. (B) Kaplan-Meier curves determined for both male (blue) and females (magenta) preBötC rhythms are significantly different from one another. The first 10 sec interburst interval was used as the endpoint metric during the transition to hypoxia.

Figure 4. Gender influences the recovery of rhythmogenesis following hypoxia.

(A) Representative traces of integrated population activity recovering from hypoxia for both a male (top) and female preBötC slice (bottom) demonstrating the stereotypical post-hypoxic depression in activity. (B) Box-whisker plots comparing TTFB between male (n = 19) and female (n = 23) rhythms.

Figure 5. Post-hypoxic recovery of rhythmogenesis correlates to f_inst prior to hypoxia in male rhythms but not in female rhythms.

(A) Representative traces of post-hypoxic recovery (i.e. TTFB) of male (left) and female (right) rhythms. Scale bar represents 2 min. Insets in each trace is the corresponding rhythm and f_inst from each experiment prior to hypoxia; scale bar represents 5 sec. (B) Linear regression analysis of f_inst prior to hypoxia to TTFB for both male (left; n = 19) and female (right; n = 23) rhythms. The r² value is greater for the male correlation and possesses a slope significantly different from 0.

Figure 6. Contribution of K_ATP channels on f_inst and TTFB.

(A) The K_ATP channel antagonist, tolbutamide (TOL, 100 to 400 µM), significantly increases f_inst of rhythmogenesis from male (n = 9) but not female (n = 9) slices and (B) eliminates the gender difference in TTFB. (C). The K_ATP channel agonist, diazoxide (60 to 100 µM), significantly decreases f_inst of rhythmogenesis from male (n = 8) and female (n = 9) slices and (D) eliminates the gender difference in TTFB.

Figure 7. Effect of glucose supplementation on f_inst and TTFB.

(A) Prior to hypoxia, glucose supplementation (30 mM) causes a significant difference in f_inst between male (n = 7) and female (n = 8) rhythms. (B) When compared to TTFB in 10 mM glucose aCSF, glucose supplementation does not affect male TTFB, but significantly reduces female TTFB.

Figure 8. Blockade of fast GABAergic and glycinergic receptors do not prevent gender difference in TTFB.

Co-application of the antagonists for fast GABAergic synaptic transmission, picrotoxin (PTX, 50 µM), and the glycineric synaptic transmission, strycnine (STR,1 µM), affects neither (A) f_inst prior to hypoxia for either gender nor (B) the gender difference in TTFB.

Figure 9. Postnatal age affects post-hypoxic recovery of rhythmogenesis.

(A) No gender differences were found in TTFB for age bins at postnatal days 2–3 (male n = 6; female n = 9) or 6–9 (male n = 9, female n = 6). (B) However, TTFB increases with age for both genders between postnatal days 10–13.