Nerve growth factor and other neurotrophins signal to neurons through the Trk family of receptor tyrosine kinases. TrkB is relatively promiscuous in vitro, acting as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4) and, to a lesser extent, NT3 (refs 3-5). Mice lacking TrkB show a more severe phenotype than mice lacking BDNF, suggesting that TrkB may act as a receptor for additional ligands in vivo. To explore this possibility, we generated mice lacking NT4 or BDNF as well as mice lacking both neurotrophins. Unlike mice lacking other Trks or neurotrophins, NT4-deficient mice are long-lived and show no obvious neurological defects. Analysis of mutant phenotypes revealed distinct neuronal populations with different neurotrophin requirements. Thus vestibular and trigeminal sensory neurons require BDNF but not NT4, whereas nodose-petrosal sensory neurons require both BDNF and NT4. Motor neurons, whose numbers are drastically reduced in mice lacking TrkB, are not affected even in mice lacking both BDNF and NT4. These results suggest that another ligand, perhaps NT3, does indeed act on TrkB in vivo.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4) act via the TrkB receptor and support survival of primary somatic and visceral sensory neurons. The major visceral sensory population, the nodose-petrosal ganglion complex (NPG), requires BDNF and NT4 for survival of a full complement of neurons, providing a unique opportunity to compare gene dosage effects between the two TrkB ligands and to explore the possibility that one ligand can compensate for loss of the other. Analysis of newborn transgenic mice lacking BDNF or NT4, or BDNF and NT4, revealed that survival of many NPG afferents is proportional to the number of functional BDNF alleles, whereas only one functional NT4 allele is required to support survival of all NT4-dependent neurons. In addition, subpopulation analysis revealed that BDNF and NT4 can compensate for the loss of the other to support a subset of dopaminergic ganglion cells. Together, these data demonstrate that the pattern of neuronal dependencies on BDNF and NT4 in vivo is far more heterogeneous than predicted from previous studies in culture. Moreover, BDNF knockout animals lack a subset of afferents involved in ventilatory control and exhibit severe respiratory abnormalities characterized by depressed and irregular breathing and reduced chemosensory drive. BDNF is therefore required for expression of normal respiratory behavior in newborn animals.

A complete understanding of the neural mechanisms responsible for the chemoreceptor and baroreceptor reflexes requires precise knowledge of the locations and chemical phenotypes of higher-order neurons within these reflex pathways. In the present study, the protein product (Fos) of the c-fos protooncogene was used as a metabolic marker to trace central neural pathways following activation of carotid sinus nerve afferent fibers. In addition, immunohistochemical double-labeling techniques were used to define the chemical phenotypes of activated neurons. Both electrical stimulation of the carotid sinus nerve and physiological stimulation of the carotid bodies by hypoxia induced Fos-like immunoreactivity in catecholaminergic neurons containing tyrosine hydroxylase or phenylethanolamine-N-methyltransferase in the ventrolateral medulla oblongata and, to a lesser degree, in the dorsal vagal complex. Tyrosine hydroxylase/Fos colocalization was also observed in the locus coeruleus and the A5 noradrenergic cell group in pons. Many serotoninergic neurons in nucleus raphe pallidus, nucleus raphe magnus, and along the ventral medullary surface contained Fos-like immunoreactivity. In pons and midbrain, Fos-like immunoreactivity was observed in the lateral parabrachial and Kölliker-Fuse nuclei, the inferior colliculus, the cuneiform nucleus, and in the vicinity of the Edinger-Westphal nucleus, but no catecholaminergic or serotoninergic colocalization was observed in these regions. Although Fos-labeled cells were observed within and lateral to the dorsal raphe nucleus, few were catecholaminergic or serotoninergic. This study further defines a potential central neuroanatomical substrate for the chemoreceptor and/or baroreceptor reflexes.

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To define the role of environmental oxygen in regulating postnatal maturation of the carotid body afferent pathway, light and electron microscopic methods were used to compare chemoafferent neurone survival and carotid body development in newborn rats reared from birth in normoxia (21 % O2) or chronic hyperoxia (60 % O2). Four weeks of chronic hyperoxia resulted in a significant 41 % decrease in the number of unmyelinated axons in the carotid sinus nerve, compared with age‐matched normoxic controls. In contrast, the number of myelinated axons was unaffected by hyperoxic exposure. Chemoafferent neurones, located in the glossopharyngeal petrosal ganglion, already exhibited degenerative changes following 1 week of hyperoxia from birth, indicating that even a relatively short hyperoxic exposure was sufficient to derange normal chemoafferent development. In contrast, no such changes were observed in the vagal nodose ganglion, demonstrating that the effect of high oxygen levels was specific to sensory neurones in the carotid body afferent pathway. Moreover, petrosal ganglion neurones were sensitive to hyperoxic exposure only during the early postnatal period. Chemoafferent degeneration in chronically hyperoxic animals was accompanied by marked hypoplasia of the carotid body. In view of previous findings from our laboratory that chemoafferent neurones require trophic support from the carotid body for survival after birth, we propose that chemoafferent degeneration following chronic hyperoxia is due specifically to the loss of target tissue in the carotid body.

Serotonin (5HT) is a powerful modulator of respiratory circuitry in vitro but its role in the development of breathing behavior in vivo is poorly understood. Here we show, using 5HT neurondeficient Pet-1 (Pet-1 −/− ) neonates, that serotonergic function is required for the normal timing of postnatal respiratory maturation. Plethysmographic recordings reveal that Pet-1 −/− mice are born with a depressed breathing frequency and a higher incidence of spontaneous and prolonged respiratory pauses relative to wild type littermates. The wild type breathing pattern stabilizes by postnatal day 4.5, while breathing remains depressed, highly irregular, and interrupted more frequently by respiratory pauses in Pet-1 −/− mice. Analysis of in vitro hypoglossal nerve discharge indicates that instabilities in the central respiratory rhythm generator contribute to the abnormal Pet-1 −/− breathing behavior. In addition, the breathing pattern in Pet-1 −/− neonates is susceptible to environmental conditions, and can be further destabilized by brief exposure to hypoxia. By postnatal day 9.5, however, breathing frequency in Pet-1 −/− animals is only slightly depressed compared to wild type, and prolonged respiratory pauses are rare, indicating that the abnormalities seen earlier in the Pet-1 −/− mice are transient. Our findings provide unexpected insight into the development of breathing behavior by demonstrating that defects in 5HT neuron development can extend and exacerbate the period of breathing instability that occurs immediately after birth during which respiratory homeostasis is vulnerable to environmental challenges.

Null mutations affecting members of the transforming growth factor-␤ and neurotrophin families result in overlapping patterns of neuronal cell death. This is particularly striking in the cranial sensory nodose-petrosal ganglion complex (NPG), in which loss of either glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4) results in a 30-50% reduction in neuronal survival. It is unknown, however, whether GDNF and any single neurotrophin support survival of the same cells, and if so, whether they are required simultaneously or sequentially during development. To approach these issues we defined survival requirements of nodose and petrosal neurons for GDNF in vitro and in bdnf, gdnf, and bdnf/gdnf null mutant mice, as well as the distribution of GDNF in NPG target tissues. Our analyses focused on the total population of ganglion cells as well as the subset of NPG neurons that are dopaminergic.Neuron losses in bdnf/gdnf double mutants are not additive of the losses in single bdnf or gdnf null mutants, indicating that many cells, including dopaminergic neurons, require both GDNF and BDNF for survival in vivo. Moreover, both factors are required during the same period of development, between embryonic day (E) 15.5 and E17.5. In addition, GDNF, like BDNF is expressed in target tissues at the time of initial target innervation and coincident with GDNF dependence of the innervating neurons. Together, these findings demonstrate that both GDNF and BDNF can act as target-derived trophic factors and are required simultaneously for survival of some primary sensory neurons. Key words: GDNF; BDNF; primary sensory neurons; growth factors; neurotrophins; knock-out mice; nodose ganglion; petrosal ganglion; carotid bodyGlial cell line-derived neurotrophic factor (GDNF) and the related proteins neurturin, persephin, and artemin, comprise a subgroup of the transforming growth factor-␤ superfamily of growth and differentiation factors. The GDNF family members signal through a receptor complex consisting of a ligand-specific glycosyl-phosphatidylinositol-linked binding molecule (GFR␣) and the membrane-spanning RET (rearranged during transfection) receptor tyrosine kinase (Rosenthal, 1999;Baloh et al., 2000). GDNF supports survival of a variety of peripheral and central neurons in vitro, including midbrain dopaminergic, spinal motor, sympathetic, parasympathetic, peripheral sensory, and enteric neurons (for review, see Unsicker et al., 1998). However, analysis of GDNF or GFR␣1 knock-out mice has revealed that a more restricted subset of neurons requires GDNF for survival in vivo Pichel et al., 1996;Sanchez et al., 1996;Cacalano et al., 1998).One of the most severely affected neuronal populations in GDNF knock-out mice is the nodose-petrosal ganglion complex (NPG) of primary cranial sensory neurons, in which 40% of cells die by birth . Interestingly, targeted disruption of the genes encoding brain-derived neurotrophic factor (BDNF) (Ernfors et al., 1994...

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