Top

Published in:

01-09-2009 | Short Communication

The efficacy of the fluorescent conjugates of cholera toxin subunit B for multiple retrograde tract tracing in the central nervous system

Authors: William L. Conte, Hiroaki Kamishina, Roger L. Reep

Published in: Brain Structure and Function | Issue 4-5/2009

Abstract

Cholera toxin subunit B (CTB) is a sensitive neuroanatomical tracer that generally transports retrogradely in the nervous system, and has been used extensively in brightfield microscopy. Recently, Alexa Fluor (AF) conjugates of CTB have been made available, which now allows multiple tracing with CTB. In this study, we examined the efficacy of these new AF-CTB conjugates when injected into the brain, and compared the results to our previous experiences using fluorescent 3k dextran amines. To test this, we injected AF 488 and AF 594 CTB into the anterior cingulate cortex and the medial agranular cortex in the rat, and examined the retrograde transport to the lateral posterior nucleus of the thalamus. We found that CTB was very viscous but yet very sensitive: small injection sites revealed very intense and detailed retrograde labeling. Anterograde transport was seen only when tissue at the injection site was damaged. These findings suggest that AF-CTB is a very reliable and sensitive retrograde tracer, and should be the first choice retrograde tracer for experiments examining multiple pathways within the same brain.

Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung C-Y, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP (2003) Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51:1699–1712PubMed

Chen S, Aston-Jones G (1995) Evidence that cholera toxin b subunit (CTB) can be avidly taken up and transported by fibers of passage. Brain Res 674:107–111PubMedCrossRef

Christianson JA, Liang R, Ustinova EE, Davis BM, Fraser MO, Pezzone MA (2007) Convergence of bladder and colon sensory innervation occurs at the primary afferent level. Pain 128:235–243PubMedCrossRef

Conte WL, Kamishina H, Corwin JV, Reep RL (2008) Topography in the projections of lateral posterior thalamus with cingulate and medial agranular cortex in relation to circuitry for directed attention and neglect. Brain Res 1240:87–95PubMedCrossRef

Conte WL, Kamishina H, Reep RL (2009) Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit b in rats. Nat Protoc 4:1157–1166PubMedCrossRef

Coolen LM, Jansen HT, Goodman RL, Wood RI, Lehman MN (1999) A new method for simultaneous demonstration of anterograde and retrograde connections in the brain: co-injections of biotinylated dextran amine and the beta subunit of cholera toxin. J Neurosci Methods 91:1–8PubMedCrossRef

Dederen P, Gribnau A, Curfs M (1994) Retrograde neuronal tracing with cholera toxin b subunit: comparison of three different visualization methods. Histochem J 26:856–862PubMedCrossRef

DeVries GH, Zetusky WJ, Zmachinski C, Calabrese VP (1981) Lipid composition of axolemma-enriched fractions from human brains. J Lipid Res 22:208–216PubMed

Fishman PH (1982) Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 69:85–97PubMedCrossRef

Kamishina H, Yurcisin GH, Corwin JV, Reep RL (2008) Striatal projections from the rat lateral posterior thalamic nucleus. Brain Res 1204:24–39PubMedCrossRef

Kamishina H, Conte WL, Patel SS, Tai RJ, Corwin JV, Reep RL (2009) Cortical connections of the rat lateral posterior thalamic nucleus. Brain Res 1264:39–56CrossRef

Kobbert C, Apps R, Bechmann I, Lanciego JL, Mey J, Thanos S (2000) Current concepts in neuroanatomical tracing. Prog Neurobiol 62:327–351PubMedCrossRef

Kreier F, Kap YS, Mettenleiter TC, van Heijningen C, van der Vliet J, Kalsbeek A, Sauerwein HP, Fliers E, Romijn JA, Buijs RM (2006) Tracing from fat tissue, liver, and pancreas: a neuroanatomical framework for the role of the brain in type 2 diabetes. Endocrinology 147:1140–1147PubMedCrossRef

Krout KE, Belzer RE, Loewy AD (2002) Brainstem projections to midline and intralaminar thalamic nuclei of the rat. J Comp Neurol 448:53–101PubMedCrossRef

Kumar RK, Chapple CC, Hunter N (1999) Improved double immunofluorescence for confocal laser scanning microscopy. J Histochem Cytochem 47:1213–1218PubMed

Llewellyn-Smith IJ, Martin CL, Arnolda LF, Minson JB (2000) Tracer-toxins: cholera toxin b-saporin as a model. J Neurosci Methods 103:83–90PubMedCrossRef

Luppi P-H, Fort P, Jouvet M (1990) Iontophoretic application of unconjugated cholera toxin b subunit (CTB) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res 534:209–224PubMedCrossRef

Masco D, Van de Walle M, Spiegel S (1991) Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates growth and differentiation of neuroblastoma N18 cells. J Neurosci 11:2443–2452PubMed

McDavid S, Lund JP, Auclair F, Kolta A (2006) Morphological and immunohistochemical characterization of interneurons within the rat trigeminal motor nucleus. Neuroscience 139:1049–1059PubMedCrossRef

Muscat L, Huberman AD, Jordan CL, Morin LP (2003) Crossed and uncrossed retinal projections to the hamster circadian system. J Comp Neurol 466:513–524PubMedCrossRef

Niedringhaus M, Jackson PG, Pearson R, Shi M, Dretchen K, Gillis RA, Sahibzada N (2008) Brainstem sites controlling the lower esophageal sphincter and crural diaphragm in the ferret: a neuroanatomical study. Auton Neurosci 144:50–60PubMedCrossRef

Panchuk-Voloshina N, Haugland RP, Bishop–Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung W-Y, Haugland RP (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem 47:1179–1188PubMed

Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates, 5th edn. Elsevier Academic Press, San Diego

van Heyningen WE (1974) Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature 249:415–417CrossRef

Wang HF, Shortland P, Park MJ, Grant G (1998) Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin b subunit, wheatgerm agglutinin and isolectin b4 from griffonia simplicifolia i in primary afferent neurons innervating the rat urinary bladder. Neuroscience 87:275–288PubMedCrossRef

Zin-Ka-Ieu S, Roger M, Arnault P (1997) Direct contacts between fibers from the ventrolateral thalamic nucleus and frontal cortical neurons projecting to the striatum: a light-microscopy study in the rat. Anat Embryol 197:77–87CrossRef

Title: The efficacy of the fluorescent conjugates of cholera toxin subunit B for multiple retrograde tract tracing in the central nervous system
Authors: William L. Conte
Hiroaki Kamishina
Roger L. Reep
Publication date: 01-09-2009
Publisher: Springer-Verlag
Published in: Brain Structure and Function / Issue 4-5/2009
Print ISSN: 1863-2653
Electronic ISSN: 1863-2661
DOI: https://doi.org/10.1007/s00429-009-0212-x

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

The efficacy of the fluorescent conjugates of cholera toxin subunit B for multiple retrograde tract tracing in the central nervous system

Abstract

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Please log in to get access to this content

Other articles of this Issue 4-5/2009

Synaptic circuitry in the retinorecipient layers of the optic tectum of the lamprey (Lampetra fluviatilis). A combined hodological, GABA and glutamate immunocytochemical study

Left cytoarchitectonic area 44 supports selection in the mental lexicon during language production

Developments of sulcal pattern and subcortical structures of the forebrain in cynomolgus monkey fetuses: 7-tesla magnetic resonance imaging provides high reproducibility of gross structural changes

Distribution of D1 and D5 dopamine receptors in the primate and rat basolateral amygdala

A volumetric study of the corpus callosum in 20s and 40s Korean people

An fMRI case study of visual memory in a patient with epilepsy: comparison before and after temporal lobe surgery