Christopher S. von Bartheld

Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system

Patterns of expression of transcripts encoding receptors for fibroblast growth factor and nerve growth factor (FGF-R and NGF-R) in the developing chick nervous system are compared using in situ hybridization histochemistry. FGF-R transcripts are expressed abundantly in the germinal neuroepithelial layer. Expression ceases as cells migrate into the mantle layer and returns during late maturation of neuronal populations, including cholinergic nuclei of the basal forebrain, brainstem reticular and motor nuclei, and cerebellar Purkinje and granule neurons. The pattern of NGF-R expression is generally reciprocal to that of FGF-R in the CNS and in some phases of development of the PNS. These results suggest that FGF and NGF may act sequentially rather than in concert during neuronal development.

Retrograde Transport of Neurotrophins from the Eye to the Brain in Chick Embryos: Roles of the p75NTR and trkB Receptors

The receptors involved in retrograde transport of neurotrophins from the retina to the isthmo-optic nucleus (ION) of chick embryos were characterized using antibodies to the p75 neurotrophin receptor and trkB receptors. Survival of neurons in the ION has been shown previously to be regulated by target-derived trophic factors with survival promoted or inhibited by ocular injection of brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF), respectively. In the present paper, we show that during the period of target dependence, these neurons express trkB and p75 neurotrophin receptor but not trkA or trkC mRNAs. We also show that BDNF and NT-3 were transported efficiently at low doses, whereas NGF was transported significantly only at higher doses. The transport of BDNF and NT-3 was reduced by high concentrations of NGF or by antibodies to either trkB or the p75 neurotrophin receptor. Thus both receptors help mediate retrograde transport of these neurotrophins. Ocular injection of the comparatively specific trk inhibitor K252a did not reduce transport of exogenous BDNF, but did induce significant neuronal death in the ION, which could not be prevented by co-injection of BDNF. Thus, transport of BDNF alone does not generate a trophic signal at the cell body when axonal trkB is inactivated. In summary, our results indicate that both p75 neurotrophin and trkB receptors can mediate internalization and retrograde transport of BDNF, but activation of trkB seems to be essential for the survival-promoting actions of this neurotrophin.

Analysis of cell death in the trochlear nucleus of the chick embryo: calibration of the optical disector counting method reveals systematic bias.

Detection of changes in numbers of neurons is essential for an understanding of neuronal development, function, and death. Optical disector counting is claimed to be the most efficient technique to estimate accurate numbers of neurons in microscopic sections. We calibrated the optical disector by comparison with three‐dimensional reconstructions from serial sections and determined how accurate this technique is relative to conventional profile counting methods. The calibration was performed on the trochlear nucleus in developing chicks. Optical disector estimates, when obtained as generally recommended, were about 25% lower than the actual number of neurons. This underestimate was caused by a nonuniform (bimodal) distribution of neuronal nuclei in paraffin and plastic (glycolmethacrylate) sections, but not in cryosections. The density of neurons in the core of the paraffin and plastic sections was substantially lower than in the upper and lower margins of these sections. Accurate estimates of neuronal numbers were obtained with a modified optical disector method that sampled the entire extent of tissue sections. Previous estimates of numbers of trochlear neurons in the developing chick have been controversial. The modified (calibrated) optical disector method revealed that the number of trochlear neurons decreased from about 1,600 at day 8.5 of incubation (embryonic day, [E]8.5) to about 900 at the time of hatching. Numbers of pyknotic nuclei peaked at E6 and at E9, revealing an additional early, but postproliferative, period of cell death. Taken together, these data emphasize the need for calibration of stereological counting techniques and the need to examine sampling strategies for potential bias. J. Comp. Neurol. 409:169–186, 1999.

Expression of BDNF and NT-3 mRNA in hair cells of the organ of Corti: quantitative analysis in developing rats.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are synthesized by inner and outer hair cells of the developing organ of Corti. This raises the possibility that the reorganization of cochlear innervation patterns that occurs postnatally may be influenced by changing levels of neurotrophin expression. To determine if differential expression of BDNF or NT-3 in the inner and outer hair cells correlates with the reorganization of afferent and efferent innervation, we used in situ hybridization techniques to quantify relative levels of transcript biosynthesis in hair cells of developing rats. BDNF transcripts decreased in inner and outer hair cells from E17 to insignificant levels at P4. NT-3 expression was high at E17 in inner and outer hair cells, decreased in outer hair cells by E21, in inner hair cells by P1, remained low during the first postnatal week and was increased in the adult. The decreases in expression of both neurotrophins at birth precede the retraction of afferent nerve terminals from outer hair cells. BDNF and NT-3 transcription decreases substantially in outer hair cells between E21 and P4 when efferent innervation begins, indicating target biosynthesis of these neurotrophins is not likely to be instrumental in efferent target selection.

Anterograde axonal transport, transcytosis, and recycling of neurotrophic factors

Traditional views of neurotrophic factor biology held that trophic factors are released from target cells, retrogradely transported along their axons, and rapidly degraded upon arrival in cell bodies. Increasing evidence indicates that several trophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), glial cell-line derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-I), and neurotrophin-3 (NT-3), can move anterogradely along axons. They can escape the degradative pathway upon internalization and are recycled for future uses. Internalized ligands can move through intermediary cells by transcytosis, presumably by endocytosis via endosomes to the Golgi system, by trafficking of the factor to dendrites or by sorting into anterograde axonal transport with subsequent release from axon terminals and uptake by second- or third-order target neurons. Such data suggest the existence of multiple “trophic currencies,” which may be used over several steps in neural networks to enable nurturing relationships between connected neurons or glial cells, not unlike currency exchanges between trading partners in the world economy. Functions of multistep transfer of trophic material through neural networks may include regulation of neuronal survival, differentiation of phenotypes and dendritic morphology, synapse plasticity, as well as excitatory neurotransmission. The molecular mechanisms of sorting, trafficking, and release of trophic factors from distinct neuronal compartments are important for an understanding of neurotrophism, but they present challenging tasks owing to the low levels of the endogeneous factors.

Positive and negative effects of neurotrophins on the isthmo-optic nucleus in chick embryos

The survival of neurons in the developing isthmo-optic nucleus (ION) is believed to depend on the retrograde transport of trophic molecules from the target, the contralateral retina. We now show that ION neurons transport nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) retrogradely and that BDNF and NT-3 support the survival of ION neurons in vivo and promote neurite outgrowth in vitro. Surprisingly, NGF enhanced normal developmental cell death in vivo in a dose-dependent way. These findings show that increased levels of NGF can have adverse effects on differentiated neurons. The negative effect of NGF could be mimicked by intraocular injection of antibodies that block binding of neurotrophins to the 75 kd neurotrophin receptor (p75). These data implicate a role for the p75 receptor in NGFs neurotoxicity and indicate that this receptor is involved in the mechanism by which ION neurons respond to BDNF and NT-3 in the target.

Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections

The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.

Synaptic targeting of retrogradely transported trophic factors in motoneurons: comparison of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and cardiotrophin-1 with tetanus toxin.

Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and cardiotrophin-1 (CT-1) are the most potent neurotrophic factors for motoneurons, but their fate after retrograde axonal transport is not known. Internalized trophic factors may be degraded, or they may be recycled and transferred to other neurons, similar to the known route of tetanus toxin. We tested whether neonatal rat hypoglossal motoneurons target retrogradely transported trophic factors to synaptic sites on their dendrites within the brainstem and subsequently transfer these trophins across the synaptic cleft to afferent synapses (transsynaptic transcytosis). Motoneurons retrogradely transport from the tongue radiolabeled GDNF, BDNF, and CT-1 as well as tetanus toxin. Quantitative autoradiographic electron microscopy showed that GDNF and BDNF were transported into motoneuron dendrites with labeling densities similar to those of tetanus toxin. Although tetanus toxin accumulated rapidly (within 8 h) at presynaptic sites, GDNF accumulated at synapses more slowly (within 15 h), and CT-1 never associated with synapses. Thus, some retrogradely transported neurotrophic factors are trafficked similarly but not identically to tetanus toxin. Both GDNF and BDNF accumulate at the external (limiting) membrane of multivesicular bodies within proximal dendrites. We conclude that tetanus toxin, GDNF, and BDNF are released from postsynaptic sites and are internalized by afferent presynaptic terminals, thus demonstrating transsynaptic transcytosis. CT-1, however, follows a strict degradation pathway after retrograde transport to the soma. Synaptic and transcytotic trafficking thus are restricted to particular neurotrophic factors such as GDNF and BDNF.

The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting.

For half a century, the human brain was believed to contain about 100 billion neurons and one trillion glial cells, with a glia:neuron ratio of 10:1. A new counting method, the isotropic fractionator, has challenged the notion that glia outnumber neurons and revived a question that was widely thought to have been resolved. The recently validated isotropic fractionator demonstrates a glia:neuron ratio of less than 1:1 and a total number of less than 100 billion glial cells in the human brain. A survey of original evidence shows that histological data always supported a 1:1 ratio of glia to neurons in the entire human brain, and a range of 40–130 billion glial cells. We review how the claim of one trillion glial cells originated, was perpetuated, and eventually refuted. We compile how numbers of neurons and glial cells in the adult human brain were reported and we examine the reasons for an erroneous consensus about the relative abundance of glial cells in human brains that persisted for half a century. Our review includes a brief history of cell counting in human brains, types of counting methods that were and are employed, ranges of previous estimates, and the current status of knowledge about the number of cells. We also discuss implications and consequences of the new insights into true numbers of glial cells in the human brain, and the promise and potential impact of the newly validated isotropic fractionator for reliable quantification of glia and neurons in neurological and psychiatric diseases. J. Comp. Neurol. 524:3865–3895, 2016.

Neurotrophic factor regulation of developing avian oculomotor neurons: differential effects of BDNF and GDNF.

Neurotrophic factors support the development of motoneurons by several possible mechanisms. Neurotrophins may act as target-derived factors or as afferent factors derived from the central nervous system (CNS) or sensory ganglia. We tested whether brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), and glial cell line-derived neurotrophic factor (GDNF) may be target-derived factors for neurons in the oculomotor (MIII) or trochlear (MIV) nucleus in chick embryos. Radio-iodinated BDNF, NT-3, NT-4, and GDNF accumulated in oculomotor neurons via retrograde axonal transport when the trophic factors were applied to the target. Systemic GDNF rescued oculomotor neurons from developmental cell death, while BDNF and NT-3 had no effect. BDNF enhanced neurite outgrowth from explants of MIII and MIV nuclei (identified by retrograde labeling in ovo with the fluorescent tracer DiI), while GDNF, NT-3, and NT-4 had no effect. The oculomotor neurons were immunoreactive for BDNF and the BDNF receptors p75(NTR) and trkB. To determine whether BDNF may be derived from its target or may act as an autocrine or paracrine factor, in situ hybridization and deprivation studies were performed. BDNF mRNA expression was detected in eye muscles, but not in CNS sources of afferent innervation to MIII, or the oculomotor complex itself. Injection of trkB fusion proteins in the eye muscle reduced BDNF immunoreactivity in the innervating motoneurons. These data indicate that BDNF trophic support for the oculomotor neurons was derived from their target.