Survival and differentiation of central noradrenergic neurons

Abstract

About half of all central noradrenergic (NA) neurons reside in the locus coeruleus (LC). Projections from the LC reach target regions throughout the entire brain and regulate a variety of functions, such as mood, vigilance, arousal, sleep, attention and memory acquisition. The development of LC noradrenergic neurons is severely affected by reductions in the availability of BMPs in vivo, and by the lack of the basic helix-loophelix protein Mash1, or the Phox2a, Phox2b or Rnx homeodomain proteins, as shown in null mutant mice.

To produce NA neurons we generated neural stem cell clones with stable overexpression of Mash1 or Phox2b and treated them with combinations of BDNF, NT-4, BMPs and FGF to induce a noradrenergic phenotype, but no TH expression was detected.

To characterize the survival requirements of developing LC neurons, we examined the involvement of rhombomere I patterning proteins. FGF8, BMP2, BMP5 and BMP7 were able to increase the number of TH positive LC neurons in E13.5 LC cultures. Of all the Writs expressed in the first rhombomere by E12.5 in mice, Wnt5a was the only one that was expressed in the vicinity of the LC and Wnt5a increased the survival of TH positive neurons in LC cultures. These results suggest that patterning signals of rhombomere I may work as survival factors in the LC at later stages of development.

Analysis of GDNF and NT-3 double null mutant mice revealed no LC abnormalities, just as in the individual knockouts. Treatment of E13.5 LC cultures with GDNF, NTN and NT-3 did not affect LC neuron survival, suggesting that these molecules are not essential for embryonic LC development. However, we show, both in vivo and in vitro, that GDNF and NTN are neuritogenic factors for LC noradrenergic neurons.

The two neurotrophins NT-4 and BDNF and their preferred receptor, TrkB, were studied for their role in LC development. We conclude that they play critical roles for the survival and phenotypic differentiation of developing LC noradrenergic neurons, possibly by linking the CREB and the Mash1-Phox2 pathways of catecholaminergic phenotype induction. TrkB is expressed in the developing mouse LC and TrkB knockout mice suffer deficient LC development. BDNF and NT-4 dramatically increase the number of TH and Phox2a immunoreactive neurons in E 13.5 rat LC primary cultures.

Examination of more mature LC neurons (E15) treated with BMP2 and/or forskolin suggested that stimulation of these cells by BMPs and cAMP plays an important role in conferring NA neurons responsiveness to several trophic factors.