Neuroanatomical tract-tracing techniques that did go viral

Fig. 1. Scheme illustrating three issues addressed in this review: (1) axonal connectivity between brain nuclei, (2) neurochemical phenotyping; 3) microcircuits inside nuclei. This scheme is repeated as ‘template’ in Figs. 2, 5, 10, and 11 to illustrate which neurons can be expected to become labeled with various classical neuroanatomical tracers, viruses, toxins, and viral vector expressions. Nucleus ‘A’ has glutamatergic (Glu) and cholinergic (ACh) neurons (first-order) projecting to nucleus ‘B’, while the projection from ‘B’ to ‘A’ comes exclusively from glutamatergic (first-order) neurons. Nucleus ‘A’ contains (second-order) GABAergic and cholinergic interneurons; nucleus ‘B’ contains (second-order) GABAergic interneurons and third-order cholinergic neurons

Fig. 2. One-dimensional neuroanatomical tracing: a anterograde tracing. Tracers such as PHA-L, BDA, RDA, Biocytin, or WGA-HRP injected in nucleus ‘A’ label complete efferent fiber trajectories including axon terminals in nucleus ‘B’. Neurons in nucleus ‘A’ are labeled irrespective of their neurochemical phenotype. In the injection site projection, neurons and interneurons both become labeled and are indistinguishable from each other. WGA-HRP and BDA are to some degree retrogradely transported (indicated here as granules in the first-order neurons in nucleus ‘B’). b Retrograde tracing. Compounds such as Fluoro-Gold, CTB, RV, and PRV label projection neurons irrespective of their neurochemical phenotype

Fig. 3. Anterograde tracing: injection sites and labeling. a, b Case 2012-6 (rat). In the same surgical session, we injected PHA-L in the central caudate putamen (CPu) of the left (L) cerebral hemisphere, while contralaterally (R), we injected BDA in the caudal CPu. Note significant retrograde transport of BDA-to-cerebral cortical pyramidal neurons ipsilateral to the injection site (dashed ellipse). Injection sites measure approximately 1 mm in diameter. c Low-power fluorescence montage in case FS-95166: injection site of PHA-L in the central nucleus (CE) of the amygdaloid complex. Bmg magnocellular basal amygdaloid nucleus. d Low-magnification montage of a combination of PHA-L tracing and neurobiotin injection. PHA-L-labeled fibers (red; Cy3) in layers II–III of medial parahippocampal cortex in contact with apical dendrites of neurobiotin-labeled pyramidal cells (green) located deep (layer V). LD lamina dissecans. e Low magnification: combination of PHA-L tracing and AF555 intracellular injection. An apical dendrite of an intracellularly AF555 injected hippocampal CA1 pyramidal neuron (red) penetrating stratum lacunosum moleculare (LM) The latter contains a terminal field of PHA-L…

Fig. 4. Two-dimensional neuroanatomical tracing. High-power confocal laser scanning images of the two-dimensional experiments illustrated in Fig. 3d, e. a Neurobiotin-labeled dendrite imaged in the green channel. b PHA-L labeled fiber (simultaneously) imaged in the infrared channel. c Merge. Encircled: an ending of a PHA-L labeled fiber (red) contacts a neurobiotin-labeled dendrite (green). d PHA-L-labeled perforant pathway fibers in stratum lacunosum moleculare imaged in the green channel, e AF555-labeled dendrite simultaneously imaged in the red channel. f Merge image; the circles indicate sites where boutons of PHA-L labeled fibers are in contact with the AF555 labeled dendrite. Images a–f are Z-projections. g 3D computer reconstruction made on basis of the images in (a, b). H, 3D computer reconstruction made on basis of the images in (d, e). Contacts are encircled

Fig. 5. Multi-dimensional neuroanatomical tracing. A layer of immunohistochemistry is added to the tracing layer to distinguish neurochemical phenotypes. Many layers are possible. a ‘Neurochemical fingerprinting’: anterograde tracing with a suitable tracer followed by immunohistochemistry using an antibody to detect a marker inside tracer-labeled axon terminals. b Immunocytochemistry to reveal the neurochemical phenotype of retrogradely labeled neurons (in this example cholinergic neurons)

Fig. 6. Multi-dimensional experiment. a Low-power image montage of the injection site of BDA in the basomedial nucleus of the amygdala (Bmg; rat LB95037). Ce central amygdaloid nucleus, LA lateral amygdaloid nucleus, PAcx periamygdaloid cortex, PIRcx piriform cortex, ot optic tract. b High-power imaging; sampling in striatum: Z-projection merge image of Z-stacks of images acquired at 488, 546, and 647 nm excitation in the confocal instrument. Color coding: green: VGluT1, red: BDA; blue: VGluT1. Boxed area: expression of VGluT1 inside a swelling (bouton) of a BDA-labeled fiber. c–e Z-projections of, respectively, the images acquired at 488 nm (VGluT1), 543 nm (BDA), and 647 nm (VGluT2). f 3D computer reconstruction. The inset is the fiber seen in the boxed area, frame (b). The BDA-labeled fiber (red) contains VGluT1 immunofluorescence (green) in both a bouton and in the fiber shaft (arrows). Notice that there is no colocalization of VGluT1 and VGluT2

Fig. 7. Multi-dimensional experiment combining in a rat dual-anterograde tracing with PHA-L (blue) and BDA (red) with retrograde tracing using rabies virus (RV; green). Here, we wanted to evaluate whether corticostriatal and thalamostriatal projections converge onto striatofugal neurons. PHA-L and BDA were iontophoretically delivered into the primary motor cortex and parafascicular thalamic nucleus, respectively. RV was pressure-delivered into either the external globus pallidus or in the substantia nigra pars compacta. a Striatopallidal; B striatonigral neuron. The insets show details. Detection of RV was carried out with an antibody against a soluble rabies phosphoprotein, resulting in Golgi-silver impregnation like labeling of dendrites, with details like dendritic spines

Fig. 8. Multi-dimensional experiment combining retrograde BDA tracing (green fluorescence) and in situ proximity ligation assay (PLA, red fluorescence) in the non-human primate brain. Here, the goal was to identify the precise location of GPCR heteromers within striatopallidal projecting neurons. For these purposes, BDA was pressure-injected into the internal division of the globus pallidus, which was further visualized with an Alexa Fluor® 488-tagged streptavidin. Next, the PLA technique was carried out for disclosing the precise location of GPCR heteromers within identified projection neurons within the post-commissural putamen. Each red dot represents one GPCR heteromer

Fig. 9. Transsynaptic retrograde tracing in rat with rabies virus (RV). a The investigated two-neuron chain schematically indicated on the right (red arrows): cerebral cortex to striatum, striatum-to-substantia nigra. The yellow arrows on the left indicate the retrograde tracing route. RV was pressure-injected into the substantia nigra. One week later, both first-order neurons (medium-sized striatal spiny striatonigral-projecting neurons) and second-order neurons (corticostriatal-projecting pyramidal neurons) could be observed. b, c Enlarged portions (the boxed areas in a). Retrograde transsynaptic spread of RV enables the accurate detection of neurons linking successively cerebral cortex, striatum, and substantia nigra. Detection of RV was with a primary antibody against a soluble rabies viral phosphoprotein, followed by a biotinylated IgG, then incubated with an HRP-tagged streptavidin and finally visualized with DAB

Fig. 10. Advanced microcircuit visualization via a helper virus injection-rabies virus tracing paradigm according to Wickersham et al. (2007b). Schematic illustration of the components in the connectivity template of Fig. 1 that can be expected to become labeled. First-order neurons accumulate GFP (green), while the second-order neurons accumulate mCherry (red)

Fig. 11. Components in the connectivity template of Fig. 1 that can be expected to become labeled when various neurotropic virus tracers and viral vectors are applied. a Classical neurotropic viruses: HSV-1, rabies virus, and swine rabies virus (PRV), retrogradely infect first-order neurons and proceed transsynaptically to second-order neurons, and so on. After several transfers, all neurons are infected and will stain. b Glycoprotein deficient virus lacks the gene that is responsible for transsynaptic transfer and, therefore, retrogradely labels only first-order neurons. c A focally injected, genetically engineered, non-replicating AAV virus infects a small population of neurons in ACh-Cre-dependent mice and inserts a reversed eYFP-coding gene in the genome of these neurons. Only neurons expressing a specific gene [in this case, the gene that codes for the choline acetyltransferase enzyme (ChAT) that synthesizes acetylcholine (ACh)] also express DNA recombinase that subsequently is responsible for ‘restoring’ eYFP expression. eYFP accumulates in all neuronal processes of these cells and acts as ‘anterogradely transported’ tracer. Cells that do not express the ChAT gene do not express DNA…

Fig. 12. Example of tracing in a genetically engineered mouse ChAT-Cre. Expression of eYFP in cholinergic neurons, switched on through transfection after injection of a modified, double-floxed EF1α–DIO–EYFP–WPRE–pA AAV2 vector (as schematically explained in Fig. 10). a Confocal laser scanning Image at low magnification of an injection spot in the diencephalon–mesencephalon transition (entopeduncular nucleus). b eYFP expressing cholinergic neuron in the boxed area of A scanned at higher magnification. All morphological details of the neuron can be appreciated