Brain-derived neurotrophic factor is required for normal development of the central respiratory rhythm in mice

Abstract

1. Molecular mechanisms underlying maturation of the central respiratory rhythm are largely unknown. Previously, we found that brain-derived neurotrophic factor (BDNF) is required for expression of normal breathing behaviour in newborn mice, raising the possibility that maturation of central respiratory output is dependent on BDNF. 2. Respiratory activity was recorded in vitro from cervical ventral roots (C1 or C4) using the isolated brainstem-spinal cord preparation from postnatal day (P) 0.5-2.0 and P4.5 wild-type mice and mice lacking functional bdnf alleles. 3. Loss of one or both bdnf alleles resulted in an approximately 50% depression of central respiratory frequency compared with wild-type controls. In addition, respiratory cycle length variability was 214% higher in bdnf null (bdnf-/-) animals compared with controls at P4.5. In contrast, respiratory burst duration was unaffected by bdnf gene mutation. 4. These derangements of central respiratory rhythm paralleled the ventilatory depression and irregular breathing characteristic of bdnf mutants in vivo, indicating that central deficits can largely account for the abnormalities in resting ventilation produced by genetic loss of BDNF. BDNF is thus the first growth factor identified that is required for normal development of the central respiratory rhythm, including the stabilization of central respiratory output that occurs after birth.

Figure 1. Influence of bdnf gene mutation on central respiratory output

A, central respiratory activity was recorded in vitro on the first (P0.5) and fifth (P4.5) postnatal day from the C4 ventral root in wild-type (+/+), bdnf heterozygous (+/-) and bdnf knockout (-/-) brainstem–spinal cord preparations. A markedly lower frequency of respiratory discharges in bdnf mutants compared with wild-type controls was apparent at both ages tested. At both ages, the preparations of different genotypes were derived from littermates. B, bulbospinal transection (arrow) abolished respiratory activity recorded from the C4 ventral root. This recording was made from the same P0.5 wild-type preparation shown in A.

Figure 2. Mutation of the bdnf gene results in decreased frequency and increased variability of respiratory discharges

Mean frequency of respiratory discharges (A) and respiratory cycle length variability (B) in wild-type (+/+), bdnf heterozygous (+/-) and bdnf knockout (-/-) in vitro brainstem–spinal cord preparations recorded during the first 48 h after birth (P0.5–2.0) and on P4.5. Respiratory cycle length variability is expressed as a coefficient of variation (s.d./mean). The mean number of measured respiratory cycles was not significantly different among groups (P0.5–2.0: +/+, 90 ± 17.4; +/−, 59 ± 8.2; −/−, 57 ± 10.3; P4.5: +/+, 120 ± 12.8; +/−, 95 ± 9.8; −/−, 105 ± 21.7). The number of preparations in each group is as follows: P0.5–2.0: +/+, 27; +/−, 57; −/−, 30; P4.5: +/+, 15; +/−, 25; −/−, 8; **P < 0.01, ***P < 0.001.

Figure 3. Maximal length of the respiratory cycle is disproportionately greater in bdnf null mutants compared with wild-type and heterozygous animals at P4.5

Mean values of maximal respiratory cycle length in P0.5–2.0 and P4.5 in vitro brainstem–spinal cord preparations (+/+ wild-type, +/−bdnf heterozygous, and −/−bdnf knockout). At P0.5–2.0 there were no statistically significant differences among genotypes, whereas at P4.5 there was a significantly longer (***P < 0.001) maximal cycle length in bdnf knockout mice. The number of preparations in each group is the same as in Fig. 2.