Endocytic and exocytic pathways of the neuronal secretory process and trans synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo

The lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) was employed to study the endocytic and exocytic pathways of the secretory process in neurons and the potential for trans-synaptic transfer of molecules within the CNS. WGA-HRP binds to surface membrane oligosaccharides and enters cells by adsorptive endocytosis. The lectin conjugate was administered intranasally or into the cerebral ventricles of mice; postinjection survival times ranged from 5 minutes to 6 days. Due to binding of the lectin to ependymal cells subsequent to an intraventricular injection, only select populations of neurons (i.e., hippocampal formation; paraventricular nuclei; midbrain raphe; VI, X, XII motor nuclei; among others) were exposed extracellularly to WGA-HRP and became labeled by retrograde axoplasmic transport from axon terminals or by direct cell body/ dendritic uptake. WGA-HRP delivered intranasally was endocytosed by first-order olfactory neurons and transported by anterograde axoplasmic flow to the terminal field within the glomerular layer of the main olfactory bulb; eventually perikarya of the mitral cell layer were labeled, presumably by anterograde trans-synaptic transfer of the lectin conjugate. In the variety of neurons analyzed ultrastructurally following exposure to WGA-HRP, the proposed sequence of intracellular pathways through which peroxidase reaction product was traced over time was: cell surface membrane → endocytic structures → endosomes (presecondary lysosomes) → transfer vesicles → transmost Golgi saccule → vesicles, vacuoles, and/or dense core granules. WGA-HRP also labeled vesicles and tubules that were channeled to and/or derived from spherical endosomes, dense bodies, and multivesicular bodies. The peroxidase-positive, membrane-delimited products of the trans Golgi saccule contributed to anterograde axonal transport vectors and accumulated within axon terminals. A second contribution to these vectors was provided by peroxidase-labeled tubules and dense bodies believed to represent components of the lysosomal compartment. Profiles of the axonal reticulum comparable to those that stained cytochemically for glucose-6-phosphatase activity, a marker for the endoplasmic reticulum, were not associated with the transport of WGA-HRP. Trans-synaptic transfer of WGA-HRP from primary olfactory neurons to postsynaptic cells in the olfactory bulb was reflected in peroxidase-positive endocytic vesicles, endosomes, dense bodies, and the trans Golgi saccule. Native HRP, which is taken into cells by fluid phase endocytosis, served as a control and was delivered into the CNS by intranasal, intravenous, or intraventricular injection. Organelles that contained native HRP were identical to those labeled with WGA-HRP, excluding the Golgi complex and its membrane-delimited products; exocytosis and trans-synaptic transfer of native HRP were not evident. The results suggest that in the neuron: (1) Macromolecules processed and packaged for export by the Golgi complex are transported independently of the axonal endoplasmic reticulum. This transport may be directed throughout the neuron and is similar to that which occurs in non-neural cells; (2) Populations of vesicles within the axon terminal are derived from the Golgi complex and/or endocytosis; and (3) WGA-HRP molecules or fragments thereof packaged within Golgi-derived vesicles, vacuoles, and dense core granules are exocytosed from axon terminals for adsortive endocytosis and possibly fluid-phase endocytosis by postsynaptic neurons. The trans-synaptic transfer of macromolecules processed and packaged in the neuron is dependent upon the Golgi complex and the exocytic/endocytic pathways of the secretory process.