Perturbed neural activity disrupts cerebral angiogenesis during a postnatal critical period

During the neonatal period, activity-dependent neural-circuit remodelling coincides with growth and refinement of the cerebral microvasculature 1,2 . Whether neural activity also influences the patterning of the vascular bed is not known. Here we show in neonatal mice, that neither reduction of sensory input through whisker trimming nor moderately increased activity by environmental enrichment affects cortical microvascular development. Unexpectedly, chronic stimulation by repetitive sounds, whisker deflection or motor activity led to a near arrest of angiogenesis in barrel, auditory and motor cortices, respectively. Chemically induced seizures also caused robust reductions in microvascular density. However, altering neural activity in adult mice did not affect the vasculature. Histological analysis and time-lapse in vivo two-photon microscopy revealed that hyperactivity did not lead to cell death or pruning of existing vessels but rather to reduced endothelial proliferation and vessel sprouting. This anti-angiogenic effect was prevented by administration of the nitric oxide synthase (NOS) inhibitor L-NAME and in mice with neuronal and inducible NOS deficiency, suggesting that excessive nitric oxide released from hyperactive interneurons and glia inhibited vessel growth. Vascular deficits persisted long after cessation of hyperstimulation, providing evidence for a critical period after which proper microvascular patterning cannot be re-established. Reduced microvascular density diminished the ability of the brain to compensate for hypoxic challenges, leading to dendritic spine loss in regions distant from capillaries. Therefore, excessive sensorimotor stimulation and repetitive neural activation during early childhood may cause lifelong deficits in microvascular reserve, which could have important consequences for brain development, function and pathology. The development of a cerebral microvascular network that precisely matches regional metabolic demands is crucial given the brain’s high energy consumption and susceptibility to ischaemia 3 . Although major cerebral vessels form during embryonic development, microvascular sprouting and pruning continue into the neonatal stages 1 , concurrent with synaptogenesis, axonal growth and gliogenesis. Common molecular pathways regulate angiogenesis and axonal growth 4 , suggesting that coordinated mechanisms establish a microvascular network that meets the requirements of adjacent neural tissue. Although some studies suggest there is a link between neural activity and microvascular plasticity 5–9 , this remains controversial and it is unclear whether neural activity regulates vascular development or if angiogenesis follows an autonomous developmental program 10 . To address this question, we examined the effects of neural activity on cerebral microvascular development in neonatal mice. First, we reduced sensory input to the barrel cortex by bilateral whisker trimming for 10 days beginning at postnatal day 15 (P15). This reduces spiking activity and metabolism 11 , and affects dendritic spine dynamics 12 in the barrel cortex. We quantified vascular branch points and total length from confocal images of various vascular markers (Supplementary Fig. 1a–e and Supplementary Video 1) and found that this manipulation did not affect vascular density in the barrel cortex (Fig. 1a and