Endocytosis and retrograde axonal traffic in motor neurons

Spinal cord motor neurons control voluntary movement by relaying messages that arrive from upper brain centres to the innervated muscles. Despite the importance of motor neurons in human health and disease, the precise control of their membrane dynamics and its effect on motor neuron homoeostasis and survival are poorly understood. In particular, the molecular basis of the co-ordination of specific endocytic events with the axonal retrograde transport pathway is largely unknown. To study these important vesicular trafficking events, we pioneered the use of atoxic fragments of tetanus and botulinum neurotoxins to follow endocytosis and retrograde axonal transport in motor neurons. These neurotoxins bind specifically to pre-synaptic nerve terminals, where they are internalized. Whereas botulinum neurotoxins remain at the neuromuscular junction, tetanus toxin is retrogradely transported along the axon to the cell body, where it is released into the intersynaptic space and is internalized by adjacent inhibitory interneurons. The high neurospecificity and the differential intracellular sorting make tetanus and botulinum neurotoxins ideal tools to study neuronal physiology. In the present review, we discuss recent developments in our understanding of the internalization and trafficking of these molecules in spinal cord motor neurons. Furthermore, we describe the development of a reliable transfection method for motor neurons based on microinjection, which will be extremely useful for dissecting further the molecular basis of membrane dynamics and axonal transport in these cells.     

Is the retrograde axonal transport of 3H-5-HT a specific process of serotoninergic neurons?

The specificity of the retrograde axonal transport of 3H-serotonin (3H-5-HT) was radioautographically studied in the afferents to the olfactory bulb (O.B.). Injections of 3H-5-HT of different concentrations (10(-2), 10(-3), 10(-4) and 10(-5) M) were performed into the O.B. of catron pretreated rats. Following injection of 3H-5-HT (10(-2) M), a cytoplasmic perikaryal labeling was observed in the bulk of afferents to the O.B. (aminergic and non-aminergic neurons). When lower concentrations of 3H-5-HT (10(-5) M) were injected into the O.B., the retrograde labeling was only seen in the raphe dorsalis (RD) serotoninergic perikarya. The specificity of the uptake-retrograde transport of 3H-5-HT seems to depend on the selectivity of uptake by nerve terminals.     

Characterization of retrograde axonal transport of antibodies in central and peripheral neurons

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.     

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16-fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.     

Multiple axonal tracing: simultaneous detection of three tracers in the same section

Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector^®VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.     

Cytofluorometric quantitation of retrograde axonal transport in single dorsal root ganglia neurons

A technique is described by which neurons from mouse dorsal root ganglia can be dispersed in single-cell suspensions suitable for quantitative cytochemical analyses. The neurons were intact as controlled by trypan blue exclusion test, and the cell size distribution of the dispersed neurons corresponded to that of untreated, intact ganglia. Horseradish peroxidase and Evans blue applied to cut sciatic nerve, were transferred by somatopetal intra-axonal transport and accumulated in corresponding dorsal root ganglia neurons. The tracers were retained during the preparation of cell suspensions. The accumulation of the fluorescent tracer Evans blue was quantitated by cytofluorometric measurements on individual neurons.     

The minimum number of neurons in the central olfactory pathway in relation to its function: a retrograde fiber tracing study.

The present study was aimed at determining the functionally essential size of the neuronal population in the central olfactory nervous system. Using conditioned rats who had learnt to avoid repellent (cycloheximide) solution by olfaction, varying degrees of injuries were made to the lateral olfactory tract, a major central olfactory pathway connecting the olfactory bulb to the olfactory cortex. After examining their olfactory ability to discriminate cycloheximide solution from water, intact bulbar projection neurons (mitral cells) with fiber connections to the olfactory cortex were quantified using a retrograde fiber tracing technique. The numbers of retrogradely labeled mitral cells from the rats with normal olfaction ranged between 20 and 92% of the control value, while those numbers from the anosmic rats ranged between 0 and 22%. We conclude that the functionally essential neuronal population is approximately one-fifth of the total in the central olfactory pathway, a presumed threshold value in terms of the ability to avoid cycloheximide solution by olfactory discrimination.     

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.     

Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons

Neurotrophin signaling is crucial for neuron growth. While the "signaling endosomes" hypothesis is one of the accepted models, the molecular machinery that drives retrograde axonal transport of TrkB signaling endosomes is largely unknown. In particular, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. Here, using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live cortical neurons, we reveal that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing the snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing mechanistic insights into the regulation of neuronal growth and survival.     

Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles

The uptake and retrograde transport of noradrenaline (NA) within the axons of sympathetic neurons was investigated in an in vitro system. Dissociated neurons from the sympathetic ganglia of newborn rats were cultured for 3-6 wk in the absence of non-neuronal cells in a culture dish divided into three chambers. These allowed separate access to the axonal networks and to their cell bodies of origin. [3H]NA (0.5 X 10(-6) M), added to the axon chambers, was taken up by the desmethylimipramine- and cocaine-sensitive neuronal amine uptake mechanisms, and a substantial part was rapidly transported retrogradely along the axons to the nerve cell bodies. This transport was blocked by vinblastine or colchicine. In contrast with the storage of [3H]NA in the axonal varicosities, which was totally prevented by reserpine (a drug that selectively inactivates the uptake of NA into adrenergic storage vesicles), the retrograde transport of [3H]NA was only slightly diminished by reserpine pretreatment. Electron microscopic localization of the NA analogue 5-hydroxydopamine (5-OHDA) indicated that mainly large dense-core vesicles (700-1,200-A diam) are the transport compartment involved. Whereas the majority of small and large vesicles lost their amine dense-core and were resistant to this drug. It, therefore, seems that these vesicles maintained the amine uptake and storage mechanisms characteristic for adrenergic vesicles, but have lost the sensitivity of their amine carrier for reserpine. The retrograde transport of NA and 5-OHDA probably reflects the return of used synaptic vesicle membrane to the cell body in a form that is distinct from the membranous cisternae and prelysosomal structures involved in the retrograde axonal transport of extracellular tracers.     

Computational neurogenetic modelling: a pathway to new discoveries in genetic neuroscience

The paper presents a methodology for using computational neurogenetic modelling (CNGM) to bring new original insights into how genes influence the dynamics of brain neural networks. CNGM is a novel computational approach to brain neural network modelling that integrates dynamic gene networks with artificial neural network model (ANN). Interaction of genes in neurons affects the dynamics of the whole ANN model through neuronal parameters, which are no longer constant but change as a function of gene expression. Through optimization of interactions within the internal gene regulatory network (GRN), initial gene/protein expression values and ANN parameters, particular target states of the neural network behaviour can be achieved, and statistics about gene interactions can be extracted. In such a way, we have obtained an abstract GRN that contains predictions about particular gene interactions in neurons for subunit genes of AMPA, GABAA and NMDA neuro-receptors. The extent of sequence conservation for 20 subunit proteins of all these receptors was analysed using standard bioinformatics multiple alignment procedures. We have observed abundance of conserved residues but the most interesting observation has been the consistent conservation of phenylalanine (F at position 269) and leucine (L at position 353) in all 20 proteins with no mutations. We hypothesise that these regions can be the basis for mutual interactions. Existing knowledge on evolutionary linkage of their protein families and analysis at molecular level indicate that the expression of these individual subunits should be coordinated, which provides the biological justification for our optimized GRN.     

Combined retrograde tracing and immunohistochemistry of trigeminal ganglion neurons projecting to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae

Rat trigeminal ganglion neurons projecting to the oral mucosa or to tooth pulps have different cell diameters and contain different chemical markers. In the present paper we examine whether trigeminal ganglion neurons sending axons to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae differ in a similar way. Retrograde tracing with fluorescent latex microspheres revealed labelled gingival and pulpal neurons in the caudal part of the trigeminal ganglion. The gingival neurons had a unimodal size distribution (peak 11 μm; range 8–14 μm) and the pulpal neurons exhibited a bimodal size distribution (peaks 12 and 25 μm; range 10–40 μm). Immunohistochemistry revealed a calcitonin gene-related peptide-like immunoreactivity in some 40% of the gingival neurons and a substance P-like immunoreactivity in 30%. Of the small pulpal neurons about 60% exhibited a calcitonin gene-related peptide-like immunoreactivity and 15% showed a substance P-like immunoreactivity. Of the large pulpal neurons some 70% exhibited a calcitonin gene-related peptide-like immunoreactivity. These neurons did not show a substance P-like immunoreactivity. In some animals a few trigeminal ganglion neurons showed a neuropeptide Y- or a vasoactive intestinal polypeptide-like immunoreactivity. Perikarya with a tyrosine hydroxylase- or a choline acetyl transferase-like immunoreactivity were not observed. We conclude that gingiva and tooth pulps in the lower jaw of T. mariae are innervated by trigeminal ganglion neurons, the cell diameters and neuropeptide contents of which differ in a pattern similar to that in the rat. Hence, this seems to represent a conserved evolutionary pattern.     

Combined immunohistochemical and retrograde tracing reveals little evidence of innervation of the rat dentate gyrus by midbrain dopamine neurons

Although the functional neuroanatomy of the midbrain dopamine (mDA) system has been well characterized, the literature regarding its capacity to innervate the hippocampal formation has been inconsistent. The lack of expression of definitive markers for dopaminergic fibers, such as the dopamine transporter, in the hippocampus has complicated studies in this area. Here we have used immunohistochemical techniques to characterize the tyrosine hydroxylase expressing fiber network in the rat hippocampus, combined with retrograde tracing from the dentate gyrus to assess the capacity for afferent innervation by mDA neurons. The results indicate that virtually all tyrosine hydroxylase fibers throughout the hippocampus are of a noradrenergic phenotype, while the overlying cortex contains both dopaminergic and noradrenergic fiber networks. Furthermore, retrograde tracing from the dentate gyrus robustly labels tyrosine hydroxylase-immunoreactive noradrenergic neurons in the locus coeruleus but not mDA neurons.