A light and electron microscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system

Abstract

The ability of horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) covalently conjugated with HRP to label retrogradely dorsal lateral geniculate nucleus (dLGN) neurons, subsequent to injections of either marker into rat striate cortex, was assessed by counting labelled neurons in the dLGN. Rats injected with either marker in concentrations ranging from 0.1 to 100 micrograms/microliter of HRP either free or coupled to WGA were perfused 24 h later and their brains incubated using the chromagen tetramethyl benzidine. At high concentrations (2-100 micrograms/microliters), comparable numbers of labelled neurons were observed in the dLGN but at low concentrations (0.1-1.0 micrograms/ microliters), WGA-HRP labelled 2-5 times more dLGN neurons than did unconjugated HRP. The sugar N-acetylglucosamine, and free WGA added in excess to WGA-HRP, abolished the retrograde labelling of dLGN neurons. In additional rats, which received striate cortex injections of 100 micrograms/microliters of either free HRP or HRP coupled to WGA, the injection site was studied with electron microscopy after survivals of 30 min to 24 h. Similar organelles in neuronal perikarya, dendrites and axons were labelled by both markers, with the exception that only rats injected with WGA-HRP had labelled GERL in some of their neurons in striate cortex. It was concluded from these studies that: (1) WGA-HRP is a more sensitive retrograde marker than free HRP at low concentrations in the rat visual system; (2) WGA-HRP binds specifically to moieties with terminal N-acetylglucosamine; and (3) WGA-HRP, but not free HRP, is localized to neuronal GERL of striate cortex subsequent to endocytosis.

Boundaries and inhibitory molecules in developing neural tissues

“…The discrete distribution of lectin binding sites in neural tissues was already noted 20 years ago (Bruckner et al, 1985;Gurd, 1977;Reeber et al, 1980;Wells and Bernstein, 1982;Wood et al, 1974) and endogenous neural lectins have been described (Barondes, 1981;Lehmann et al, 1990). The fact that certain lectins are taken up by subsets of synaptic terminals and transported back to the neuronal cell body has been exploited for neuronanatomical tracing studies (Gerfen and Sawchenko, 1984;Schwab et al, 1978;Steindler and Bradley, 1983;Trojanowski et al, 1981). A comparison of lectin dissemination after injection of peroxidaseor distribution of WGA binding sites in the adult mouse cerebellum documented a pronounced lectin-binding matrix that outlined neurons and synaptic complexes (Steindler and Cooper, 1986).…”

“…The fact that certain lectins are taken up by subsets of synaptic terminals and transported back to the neuronal cell body has been exploited for neuronanatomical tracing studies (Gerfen and Sawchenko, 1984;Schwab et al, 1978;Steindler and Bradley, 1983;Trojanowski et al, 1981). A comparison of lectin dissemination after injection of peroxidase-or distribution of WGA binding sites in the adult mouse cerebellum documented a pronounced lectin-binding matrix that outlined neurons and synaptic complexes (Steindler and Cooper, 1986).…”

Identification of Eye-Specific Domains and Their Relation to Callosal Connections in Primary Visual Cortex of Long Evans Rats

“…Callosal connections were labeled following multiple intracortical injections of either horseradish peroxidase (HRP, Sigma Co, 25% in saline; total volume = 4.0 μL), biotinylated biodextran amine (BDA, 10 kDa, 10% in 0.9% saline, Molecular Probes, Eugene, Oregon, total volume = 4 μL), or rhodamine-tagged latex beads (RB; LumaFlour, Naples, FL, concentrated stock solution, total volume = 4 μL). Previous studies at both light and electron microscopic levels have demonstrated that HRP is transported both anterogradely and retrogradely (Trojanowski et al 1981), while BDA (10 kDa) is transported mainly anterogradely, although some retrograde labeling may occur (Reiner et al 2000). The fluorescent tracer RB is transported retrogradely (Katz et al 1984).…”

Influence of ocular dominance columns and patchy callosal connections on binocularity in lateral striate cortex: Long Evans versus albino rats

“…Injections were evenly spaced (0.2 μL each), at a depth of~800-1,000 μm. Previous studies at both light and electron microscopic levels have demonstrated that HRP is transported both anterogradely and retrogradely (Trojanowski, Gonatas, & Gonatas, 1981). HRP was pressure injected through glass micropipettes (50-100 μm tip diameter) over an area extending from~2.0 to 7.0 mm lateral to the midline suture, and 0.0-6.0 mm anterior to lambda suture.…”