Improved retrograde axonal transport and subsequent visualization of tetramethylrhodamine (TMR) -dextran amine by means of an acidic injection vehicle and antibodies against TMR.
We studied the ability of various dextran amines (DA) to retrogradely label cortical neurons to the full extent of their dendritic configurations. Corticothalamic neurons were labeled by pressure injection of DA into the ventrobasal thalamic nuclei of the rat brain. Of fluorescein-, Texas Red-, Cascade Blue- and tetramethylrhodamine (TMR)-DAs of MW 3000 and TMR-DA of MW 10,000, neurons were most efficiently labeled with TMR-DA of MW 3000. The use of acidic vehicles (pH 1-3) for dissolving TMR-DA enhanced the retrograde labeling, as compared with that of a neutral vehicle. The retrograde labeling with TMR-DA was more clearly demonstrated by using anti-TMR antibodies; the indirect immunofluorescence method with a rhodamine-conjugated secondary antibody and immunoperoxidase method with a peroxidase anti-peroxidase (PAP) complex revealed that the dendrites of many corticothalamic neurons were filled with TMR-DA. The Golgi-like retrograde labeling of TMR-DA visualized by the PAP immunoperoxidase method was comparable with that of biotinylated DA by the avidin-biotinylated peroxidase complex method. Similar Golgi-like dendritic staining was observed among corticospinal neurons after injection of TMR-DA into the corticospinal tract of the spinal cord. Most apical dendrites of corticospinal neurons extended into layer I, whereas those of corticothalamic neurons ended in layer IV or the deep part of layer III. The TMR-DA injection under acidic conditions and immunostaining with the anti-TMR antibodies are considered to be a useful method to visualize the dendrite configuration of cortical projection neurons.
Kaneko
T
T
Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Japan.
Saeki
K
K
Lee
T
T
Mizuno
N
N
D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D001923:Brain Chemistry / Q000502:physiology; D002540:Cerebral Cortex / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D003712:Dendrites / Q000502:physiology / Q000648:ultrastructure; D003911:Dextrans; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods*; D008845:Microinjections; D009434:Neural Pathways / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure*; D014677:Pharmaceutical Vehicles; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines; D013116:Spinal Cord / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D013788:Thalamus / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure
D003911:Dextrans; D005456:Fluorescent Dyes; C067661:Fluoro-Ruby; D014677:Pharmaceutical Vehicles; D012235:Rhodamines