Neuroscience Vol. 22, No. 3, pp. 1135-1144, 1987 Printed in Great Britain 0306-4522/87 $3.00 + 0.00 Pcrgamon Journals Ltd 0 1987 IBRO UPTAKE AND AXONAL TRANSPORT OF HORSERADISH PEROXIDASE ISOENZYMES BY DIFFERENT NEURONAL TYPES B. KEY* and P. P. GIORGI~ Neuroembryology Laboratory, School of Anatomy, University of Queensland, Brisbane, 4067, Australia Ahdraet-The uptake and transport of basic and acidic horseradish pcroxidase isoenxymes was compared in the neuromuscular, visual and olfatory systems of Xenepus larvae and postmetamorphic frogs. The concentration (w/v) of the two preparations was cormcted to compensate for their difference in enxymatic activity (unit/w), which was seven-fold higher in basic horseradish peroxidase. Uptake and transport of horseradish peroxidase isoenxymes could be demonstrated with 7% basic horseradish peroxidase, but not with equal amounts of 49% acidic horseradish peroxidase in all systems investigated: retrograde transport from terminals of retinal ganglion cells, isthmotectal neurons and spinal motoneurons, as well as anterograde transganglionic transport along olfactory neurons. A very weak labelling of the same neuronal pathways by acidic horseradish peroxidase was obtained only after incmasing the amount injected by approximately two-fold. Basic horseradii peroxidase isocnzymcs were also preferentially taken up and transported retrogradely by broken axons of the optic nerve. When tested, similar rc5ults were obtained in both larvae and frogs suggesting that preferential uptake and transport of basic horseradish peroxidase is a general feature of all neurons and of all developmental stages. Electron microscopical analysis of the outer layers of the optic tectum revealed that, in the same experimental conditions producing no retrotrade labelling of optic axons, acidic horseradish peroxidase was rarely found to enter nerve terminals. It appears that interactions between horseradish peroxidase and neuronal membranes occur during uptake and transport and that molecular chargeplays an importantrole, beyond non-specific fluid-phase endocytosis. We suggest that differences between horseradish peroxidase isoenqmes as neuronal tracers reflect a process of adsorptive endocytosis related to general characteristics of neuronal membranes (regardless of age) and not to specificreceptor-mediated interactions characteristic of neuronalspecificity. Nerve cells are able to endocytose certain exogenous molecules in their micro-environment, package them into membrane-bound organelles and subsequently transport them along their axons. Horseradish per- ox&se (HRP) is being used extensively to trace neuronal pathways. Although we know something about the sulubcellular compartments involved in the transport of HRP much less is understood about the mechanisms involved in the endocytosis of HRP by neurons. It was originally suggested that HRP entered neur- ons via the fluid-phase endocytosis occurring during the normal turnover of synaptic vesicles.‘Jo21~~*39 In non-neuronal cells fluid-phase endocytosis involves the invagination of the plasma membrane and the subsequent intracellular budding off of a membrane vesicle, without any interaction between HRP and the membrane. 1**37*3* Subsequent studies suggested that some form of specific HRP-membrane interaction occurs during endocytosis. When compared to acidic isoenxymes, basic isoenxymes of HRP appeared to be exclusively endocytosed and retrogradely transported by retinal ganglion cells in both rat’ and Xenopus larvae.“j The *Presentaddress:Division of Brain Research, Institute of Dcvel~pmental Research, Children’s Hospital Medical Center, Cincinnati, OH 45229, U.S.A. tAutbor to whom correspondence should be addressed. Abbreuip~ion: HRF’, horseradish peroxidase. results of subsequent in zyxwvutsrqponmlkjihgfedcbaZ vitroH and in vivo9” studies were consistent with the preferential uptake and transport of basic HRP isoenxymes. The aim of this work was to establish whether-in Xenoplrs a preferential uptake and transport of basic isoenxymes is a general feature of several neuronal types (Fig. 1) and whether this phenomenon is consis- tent at different developmental stages. At the same time we intended to provide information on HRP tracing technique in non-mammalian species. EXPERIMENTAL PROCEDURES Preparationof isoenzymes Sigma type I HRP was used to separate basic and acidic isoenxymes by ion-exchange chromatography using diethylaminoethyl-cellulose. (Sigma C&m&al CO.).‘~Acidic and basic fractions were dialysed against several changes of deionixed water for 12 h at 4°C and then freeze dried. These fractions were resolved by polyacryhrmide gel isoelectric focusing.@ The protein content of the ’ tsoenqme fractions was determined” using bovine serum albumin as standard. The enxymatic activities were measured with Odiam&hmM with the following modifications: Odianisidine (0.1%) and hydrogen peroxide (0.003%) in 0.05M potassium phos- phate bulfer (PH 7.0) as substrates in a 3.0 ml incubation mixture. The rate of production of oxidixed Odiani&ne was followed at 460 MI during a l-3 min period. Through- out this period the reaction plot was linear and the en- xymatic activity was recorded as AA&mm x pg protein. Animals Xenopus la&r tadpoles between stages 52 and 58’O and postmetamorphic Xenopu.t frogs (2 weeks) were anaes- 1135