Fast Blue

Injections were made using an image-guided stereotaxic system (Brainsight Frameless, Rogue Research, Montreal, Canada). The target area was identified on the monkey’s magnetic resonance imaging (MRI) using sulcal landmarks in a 3D reconstruction of the monkey brain and a coronal, parasagittal, or horizontal plane (Frey et al., 2004 (link)). The Brainsight system monitors injection position online and to within a few millimeters range. Injections of the fluorescent Fast blue and Diamidino yellow tracers (0.2–0.3 μl) spanning the full depth of the cortex were made into V1, V2, V4, TEO, TEpd, MT, 7a, STPc, DP, 8m, and 8L. Injection sites can be viewed in Markov et al. (2013 ).
The spatial extent of labeling and the percentages of double-labeled neurons in supragranular vs. infragranular layers (in V2, V3, MT, TEO, and TE) were computed after paired parallel longitudinal injection of 3–5 μl of the two tracers in V1 in one brain and in V4 in another brain. These paired injections, 2–3 mm apart, were used to quantify the divergence of terminal arbors and the degree of scatter in projection topology, and were made at a shallow angle to the cortical surface spanning the entire thickness of the cortical sheet. The tracer was injected while the Hamilton microsyringe was withdrawn from the cortex so as to form parallel longitudinal injection sites restricted to the cortical gray matter.
In order to quantify the frequency of single neurons sending projections to both V1 and V4, simultaneous injections were made in these two areas. In one animal, massive injections were made by multiple injection of Diamidino yellow in the opercular part of V1 and, in the same hemisphere, Fast blue was massively injected in V4 between the lunate sulcus and the superior temporal sulcus. Both sets of injections involved corresponding regions representing the lower part of the central visual field (Gattass et al., 1987 (link), 1988 (link)).
Following a 10–13 day survival period, to allow retrograde transport of the tracers, the animals were deeply anesthetized and perfused through the heart with 2.7% saline, followed by 4–8% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4), and 10–30% sucrose in PB. The brains were then blocked in the coronal, sagittal, or horizontal plane, and 40-μm-thick sections were cut on a freezing microtome. One in three sections was immediately mounted from saline solution onto 3% gelatin-coated slides. Selected sections at regular intervals from those not used for counting were reacted for cytochrome oxidase, acetylcholinesterase (AChE) activity (Barone et al., 2000 (link)), and SMI-32 (Hof et al., 1996 (link)). Sections were observed with a Leitz or Leica DMRE fluorescence microscope equipped with a D-filter set (355–425 nm). A computer-assisted program (ExploraNova) was used with a motorized microscope stage so as to trace out sections electronically and record neuron positions with high precision (±10 μm).

The sections were left without coverslips and were observed with oil-immersion objectives under UV light with a fluorescent microscope equipped with a D-filter set (355–425 nm). The characteristics of neurons labeled with Fast blue and Diamidino yellow have been described elsewhere (Keizer et al., 1983 (link)): neurons labeled by Fast blue exhibit a blue coloration in their cytoplasm while those labeled by Diamidino yellow exhibit a yellow nucleus. After plotting, sections were counterstained for Nissl substance and backprojected onto the charts of labeled neurons so as to trace cytoarchitectonic areal and laminar borders.
Accurate estimates were made of the numbers of neurons per area with respect to the total number of neurons encountered in one cortical hemisphere by plotting 1/3 sections throughout the brain (Vezoli et al., 2004 (link)). The fraction was expressed as the FLN (fraction of labeled neurons) and the percentage of SLN (Fig. 1) (Barone et al., 2000 (link)).
SLN values and distances are listed in Table 2. FLN values are published in Markov et al. (2013 ). Tracer injection leads to dense labeling of extensive regions of the cortex. The full set of source areas projecting to each of our injected target areas is reported elsewhere (Markov et al., 2012 ). The present study used the totality of labeled neurons to estimate the FLN values but restricted the list of source areas considered to those that have their analog in the FVE hierarchical model. The source areas reported in this study include V1, V2, V3, V3A, V4, V4t, 7A, 7B, LIP, STPr, STPi, STPc, FST, MST, MT, TEpd, TEpv, TEad, TEav, TEa/ma, TEa/mp, 8L, 8m, TEO, TEOm, DP, V6, V6A, VIP, PIP, TF, TH, MIP, 7m, 9/46d, 9/46v, 46v, 46d, perirhinal (Peri), and entorhinal (Ento). A full description of atlases can be found at http://www.core-nets.org. Multiple criteria were used to allocate labeled neurons to particular extrastriate areas, as described elsewhere (Hof and Morrison, 1995 (link); Stepniewska and Kaas, 1996 (link); Kaas and Hackett, 1998 (link); Barone et al., 2000 (link); Falchier et al., 2002 (link); Clavagnier et al., 2004 (link); Markov et al., 2011 (link), 2012 ).
A central aspect of the present study is the laminar location of parent neurons of FF and FB pathways. In area V1, labeled neurons in layer 4B were classified as SLNs. In extrastriate cortex two distinct compartments were distinguished in the supragranular layers: layers 2/3A and 3B (Fig. 2).

For tracing experiments, animals in which Fast Blue injection was circumscribed to the right medulla including the gigantocellular reticular nucleus (GI ), the lateral paragigantocellular reticular nucleus (LPGi), the alpha and ventral pars of the gigantocellular reticular nucleus (GiV/α) we included for analysis. Every two slices were imaged using an epifluorescence microscope (Olympus BX51, Tokyo, Japan) associated with stereology software (Stereo Investigator, MicroBrightField Bioscience, Colchester, VT). Using anatomical landmarks, the borders of the CnF and PPN were traced. A random subsampling was used, and neurons were counted in selected sections (50–150 μm² squares). Sizes of the sections were determined by the surface on the slices and the volume of the nucleus. Taking in consideration the number of counted cells, the interval between sections, the area of the nucleus, and the thickness of the slices, the number of counted cells was estimated. In some experiments, all neurons were counted manually every other section enabling a 3D representation of the PPN and CnF (Figure 2C). Custom MATLAB scripts enabled pooling all mice together and representing the spatial distribution (medio-lateral and dorsoventral) of retrogradely labeled neurons.

10 adult VGlut2-Cre mice were exposed to a laminectomy and 16 VGlut2-Cre mice to a laminectomy followed by a lateral thoracic hemisection. 8 weeks after SCI or sham surgery, under isoflurane (1.5%–2% O2) anesthesia, the mouse was installed in a stereotaxic frame; a craniotomy was performed for retrograde tracer injection: Fast Blue (Polysciences, 17,740-5, 50 nL, 2% suspension in phosphate buffer with 2% dimethyl sulfoxide) in the contralesional medulla: AP = 5.5 mm; L = 0.5 mm; D = 5.5 mm. A 2 μL Hamilton neuro syringe was used to perform this injection. To avoid leakage, the neuro syringe was kept in place for 2 min and removed slowly after the injection. Mice were perfused 4 days after the Fast Blue injection. Mice with SCI extending over the other side of the spinal cord and Fast Blue injections leaking in the ipsilesional brainstem were excluded from our analysis. We analyzed a total of 5 SCI and 5 sham mice for our tracing experiments.

Surgical procedures were conducted in the morning to mid-afternoon in WSU’s LAR sterile surgical suite, and each animal underwent one surgery. Mice were anesthetized with isoflurane at 3–5% for induction, then maintained at (2–3%) during surgery via nose cones. Four hindlimb muscles—soleus (Sol), tibialis anterior (TA), and lateral and medial gastrocnemius (LG and MG)—were exposed by a small incision and separation of the overlying biceps femoris muscle. In a given mouse, all four muscles were injected with one of the following tracer/concentration protocols: (1) 5 µL of Fast-Blue (FB) (Polyscience, Warrington, PA, USA catalog 17740-1) at (i) 0.1%, (ii) 0.2%, or (ii) 2%; in weight/volume; or (2) 5 µL of CTB-488 Alexa Fluor conjugate (Invitrogen, Waltham, MA, USA catalog C22841) at (i) 0.05% or (ii) 0.1%; in weight/volume. Injections were given through a 10 μL Hamilton syringe with a 33-gauge needle. Overall, each mouse received a total of 20 µL of one type/concentration of tracer injected into its four hindlimb muscles. Intraoperative monitoring was conducted every 5 min based on movement, respiration, and color. Buprenorphine (0.0025 mL/g) was injected subcutaneously immediately after surgery followed by subcutaneous injection of Carprofen (0.01 mL/g) 24 h after surgery for post-operative pain relief. Mice were then euthanized and perfused 3- or 5-days post-injection of tracers (labeling duration).