Several conclusions can be drawn from the present work. First, BDNF is essential for normal development of multiple cell types in the nervous system, including neurons in multiple sensory ganglia, the cerebral cortex, the hippocampus, and the striatum. Second, BDNF is not essential for all functions suggested by previous observations. For example, analysis of the BDNFneo homozygotes suggests that BDNF is not essential for survival of motor neurons, cerebellar granule cells, dopaminergic neurons in the substantia nigra, cholinergic neurons in the basal forebrain, or retinal ganglion cells (latter not shown). Finally, our data regarding reductions in neuropeptide and calcium-binding protein expression suggest that BDNF regulates postnatal brain differentiation, as suggested previously by BDNF and trkB mRNA expression studies.
As shown above, disruption of the BDNF gene results in a profound loss of sensory neurons. Sensory neuron populations derived from ectodermal placodes are particularly affected, consistent with previous observations on the activity of BDNF on a large proportion of these neurons in primary culture (reviewed by Korsching, 1993
). Cell losses in the vestibular ganglion were particularly striking and are consistent with previous observations concerning BDNF expression in the sensory epithelia innervated by vestibular neurons and the responsiveness of these cells to BDNF in vitro (see, e.g., Pirvola et al., 1992
). Vestibular deficits are likely to explain in part the abnormal behavior of BDNFneo
mutant mice, including the observed difficulty in remaining upright, in ataxia, and in spinning. The cell losses in the petrosal and nodose ganglia could explain the lethality of this mutation. Sensory neurons in these ganglia relay information from the heart, lungs, great vessels, and gut to the central nervous system. This sensory information is used in regulation of heart rate, blood pressure, respiratory rate, bronchodilation, gut motility, and many other aspects of visceral function. The observed deficits in cranial and dorsal root ganglia are also likely to affect the viability of the mutant. Sensory neurons in these ganglia are extraordinarily diverse, differing in sensory modality and many other properties (see Scott, 1992
). Our studies suggest that muscle spindle afferents are unaffected in the BDNPneo
mutant but that mechanoreceptors are likely to be affected.
Effects of BDNF addition on motor neurons in vitro and in vivo have suggested strongly that BDNF is a physiological regulator of their survival and development (see Korsching, 1993
). Many fewer motor neurons are present in mice lacking the BDNF receptor TrkB (Klein et al., 1993
). Despite these suggestive precedents, in homozygous BDNPneo
mice, all examined populations of motor neurons appear to be present in normal numbers and to have differentiated normally by several criteria. Similarly, the BDNPneo
mutant does not have the substantial morphological abnormalities in brain structures that might have been predicted from earlier data. Among many possibilities, NT-3 or NT-4/NT-5, both of which have been shown to activate the TrkB BDNF receptor (see Ip et al., 1993b
), may compensate for the loss of BDNF.
The observed reductions in neuropeptide and calcium-binding protein expression in specific regions of the BDNPneo mutant brain suggest that BDNF may directly regulate the differentiation of many brain neurons. Expression of NPY was normal in one forebrain structure, the striatum, but reduced in cerebral cortex and hippocampus of 15-, 17-, and 20-day-old BDNPneo mutants and did not appear to rise significantly in the older animals, suggesting that maturation of neuropeptide expression is not simply delayed, but is permanently affected.
Calcium-binding proteins may be required in normal quantities for normal neuronal functioning since intracellular calcium regulates many aspects of neuronal metabolism (Baimbridge et al., 1992
). The deficiency in calbindin expression in the BDNPneo
mutant striatum may result in abnormal function in the striatonigral pathway, which could account for some of the observed abnormal motor behaviors. Parvalbumin expression was specifically reduced in the cerebral cortex (with especially severe region-specific reductions) and in the hippocampus of BDNFneo
mutants. Parvalbumin is localized to a subset of GABAergic neurons in the cerebral cortex and hippocampus. In hippocampus, expression levels correlate with high metabolic and firing rates (see Baimbridge et al., 1992
). If, as seems likely, parvalbumin modulates GABAergic neuron function by buffering calcium, the observed deficits may prevent these neurons from functioning normally.
In conclusion, the phenotype of the BDNFneo mutant mouse demonstrates that this factor is absolutely required for development of some peripheral sensory neurons. In contrast, requirements for BDNF may be more modest in the brain. Anterior brain structures have been added progressively in vertebrate evolution. The phenotype of the BDNFneo mutant suggests that the events underlying differentiation of the brain involve more subtlety than those regulating more ancient parts of the vertebrate nervous system.