Eliminating medullary 5-HT neurons delays arousal and decreases the respiratory response to repeated episodes of hypoxia in neonatal rat pups

Abstract

Arousal from sleep is a critical defense mechanism when infants are exposed to hypoxia, and an arousal deficit has been postulated as contributing to the etiology of the sudden infant death syndrome (SIDS). The brainstems of SIDS infants are deficient in serotonin (5-HT) and tryptophan hydroxylase (TPH) and have decreased binding to 5-HT receptors. This study explores a possible connection between medullary 5-HT neuronal activity and arousal from sleep in response to hypoxia. Medullary raphe 5-HT neurons were eliminated from neonatal rat pups with intracisterna magna (CM) injections of 5,7-dihydroxytryptamine (DHT) at P2-P3. Each pup was then exposed to four episodes of hypoxia during sleep at three developmental ages (P5, P15, and P25) to produce an arousal response. Arousal, heart rate, and respiratory rate responses of DHT-injected pups were compared with pups that received CM artificial cerebrospinal fluid (aCSF) and those that received DHT but did not have a significant reduction in medullary 5-HT neurons. During each hypoxia exposure, the time to arousal from the onset of hypoxia (latency) was measured together with continuous measurements of heart and respiratory rates, oxyhemoglobin saturation, and chamber oxygen concentration. DHT-injected pups with significant losses of medullary 5-HT neurons exhibited significantly longer arousal latencies and decreased respiratory rate responses to hypoxia compared with controls. These results support the hypothesis that in newborn and young rat pups, 5-HT neurons located in the medullary raphe contribute to the arousal response to hypoxia. Thus alterations medullary 5-HT mechanisms might contribute to an arousal deficit and contribute to death in SIDS infants.

Keywords: arousal; medulla; response to hypoxia; rodent development; serotonin.

Fig. 1.

A–E: 5-HT neurons at five different levels through the hindbrain and medulla illustrated schematically with corresponding coordinates relative to Bregma. Characteristic distribution of 5-HT neurons in artificial cerebrospinal fluid (aCSF) (A1–E1), injection control (IC) (A2–E2), and 5,7-dihydroxytryptamine (DHT) (A3–E3) rat pups. Areas that appeared profoundly or partially impacted by the lesion are represented by the black and dark gray zones, respectively (A1–A3). In all groups, densely packed neurons immunolabeled for tryptophan hydroxylase (TPH) were visible in the dorsal and ventral parts of the dorsal raphe nucleus (DR) (B1–B3). At the rostral pole of the raphe magnus (RM) in aCSF and IC groups, TPH-immunolabeled cells were visible in a dispersed pattern (B3). In DHT pups many fewer cells were visible (arrows) (C1–C3). Compared with aCSF and IC pups (C1 and C2), DHT pups (C3) show a profound loss of neurons within RM (triangular area indicated in C3 dorsal to the pyramids). In contrast, 5-HT neurons in raphe pallidus (single arrowhead) are visible both in aCSF and DHT cases (D1–D3). In comparison to aCSF (D1) and IC (D2), in DHT pups (D3) there was a loss of cells in RM (single arrow) and as well laterally in the paragigantocellularis lateralis (double arrows), although TPH immunoreactivity persisted to some extent in raphe pallidus (single arrowhead) (E1–E3). At the level of raphe obscurus, there were fewer 5-HT neurons and/or neurons with abnormal morphology in DHT pups; boxed region shown at higher magnification in inset. A1–A3 same scale and B–E same scale; bars in A1 and E1 = 250 microns.

Fig. 2.

Quantitative counts of TPH immunoreactive (TPH+) neurons in the RM. A: scatter graph showing the total cell counts in the aCSF (○), DHT (○) and IC (△) pup brains. B: bar graph showing the average cell counts for the three groups. The mean number of TPH+ cells in the DHT group was significantly less than in the aCSF or lesion control groups (*P < 0.001). Values are expressed as means ± SD.

Fig. 3.

Arousal latencies for the aCSF, IC, and historical control (HC) groups. A: mean arousal latencies, averaged over all trials for P5, P15, and P25 pups. B: arousal latencies for the three ages over four hypoxia trials for the three control groups. Pups in the HC group were not exposed to surgery, injections, or desipramine. There were no significant differences overall or at any age. Data are shown as means ± SE.

Fig. 4.

Mean arousal latencies, averaged across hypoxia trials by age. Latencies of pups injected with DHT (black bars) were compared with those in pups injected with aCSF (white bars with no hatching) and those injected with DHT but without a significant reduction of medullary 5-HT neurons (IC group) (white bar with course hatching). There was an overall (across all ages and hypoxia trials) lengthening of arousal latency associated with a reduction in the number of medullary 5-HT neurons. As shown in the first group of bars (MAIN), overall, arousal latencies were longer in the DHT group compared with the IC (*P = 0.008) and the aCSF Control group (*P = 0.034). The effect of 5-HT neuronal loss was most pronounced at P5 and P25. At P5 arousal latencies were longer in the DHT group compared with the IC group (#P < 0.001) and the aCSF group (#P = 0.011). At P25 arousal latencies were longer in the DHT group compared with the IC group ($P = 0.021) and the aCSF control group ($P = 0.035). At P15, arousal latencies were also longer in the DHT group compared with the aCSF and IC group, but the differences did not reach significance after correcting for multiple comparisons. Values are indicated as the mean ± SE.

Fig. 5.

A: arousal latencies across four repeated hypoxia trials at P5, P15, and P25. Pups were exposed to four episodes of hypoxia (10% O₂) begun in quiet sleep and the latency to subsequent behavioral arousal determined. Latencies of pups with loss of 5-HT neurons after DHT administration (●) were compared with those in pups injected with aCSF (○) and those injected with DHT but without a significant reduction of medullary 5-HT neurons (△). Latency increased with successive trials (habituation) in all treatment groups (P < 0.001). The mean arousal latency was greatest in the DHT pups at P5 and P25 (see Fig. 4). Values are expressed as the mean ± SE. B: Cox regression analysis showing the proportion of pups awake at various times after the onset of hypoxia. For any given time, the probability of being awake was lower in the DHT groups compared with the controls at all ages. For example, at P25, 40 s after the onset of hypoxia, 60–75% of the control pups had aroused but only 33% of the DHT-treated pups. Consistent with our mixed model analysis, the effects of DHT were more prominent at P5 (*P < 0.001) and P25 (*P < 0.001) compared with P15 (#P = 0.039).

Fig. 6.

A: mean prehypoxia respiratory frequency (f_R), averaged across trials and ages (first group of bars, Main), and across trials at P5, P15, and P25 for the DHT, aCSF, and IC treatment groups. The mean f_R was lower at P5 compared with P15 and P25 (*P < 0.001) in the IC and DHT groups, and there was no effect of treatment on f_R at any age. B: mean slope of f_R from the onset of hypoxia to arousal averaged across ages and trials (first group of bars, Main) and at P5, P15, and P25 for the DHT, aCSF, and IC treatment groups. Overall, the slope of f_R was lower in the DHT pups compared with the aCSF (*P = 0.002) and IC (P = 0.005) pups. The f_R slope was lower in DHT than the aCSF or IC pups at both P5 (#P < 0.001 vs. both aCSF and IC) and P15 ($P < 0.001 vs. both aCSF and IC). The f_R slope was also lower in the DHT compared with the aCSF pups at P25, but this did not reach significance (P = 0.053). C: the relationship between the slope of the f_R response during hypoxia and arousal latency. Control pups are shown as ○ and DHT pups as ●. Note that there is generally an inverse relationship between slope and arousal latency with no effect of DHT treatment. The regression coefficient for the control pups was −3.339, P < 0.001, and for the DHT pups, −4.597, P = 0.0057. Values in the bar graphs are expressed as the means ± SE.

Fig. 7.

A: mean prehypoxia heart rate (f_H) averaged across all ages and hypoxia trials and across all trials for P5, P15, and P25. Overall, f_H increased with age in all treatment groups. This was especially the case in the IC group where baseline f_H was significantly higher than the aCSF (*P = 0.003) and the DHT (*P < 0.001) group at P25. Although the mean f_H was higher in the IC group compared with the other two groups at P5 and P15, the differences did not reach significance. Values are expressed as the means ± SE. B: slopes of the changes in f_H from the onset of hypoxia to arousal. Overall, similar to f_R, the slope of f_H increased with age. Averaged across all ages and hypoxia trials, the f_H slope was slightly lower in the IC group compared with the aCSF (P = 0.022) and DHT (P = 0.034) groups. This was largely because at P25 the slope of the f_H response was negative and significantly different from the slopes in the two control groups (*P < 0.001). In addition, at P15 the f_H slope was lower in the DHT pups compared with the aCSF pups (P = 0.028). Values are expressed as the means ± SE.

Fig. 8.

Prehypoxia body temperature (T_B) across the four hypoxia trials at P5 for the three treatment groups. The increase in T_B across trials was significantly less in the DHT and IC groups compared with the aCSF control group. By hypoxia trial 4, T_B in the aCSF group was significantly higher than in the IC (*P = 0.020) or the DHT (*P = 0.034) group. Values are expressed as the means ± SE.