Enhancement at the injection site was largely cleared by 2 months postinjection (Figure S4C, red line), similar to the time course of the transported compound in VPL (Figure 4B). Thus, in both the injection site and the transport targets, the time course of GdDOTA-CTB enhancement was fully consistent with neuronal uptake of the selleck inhibitor tracer CTB; presumably extracellular diffusion of GdDOTA alone would clear much faster. To confirm the latter, we directly measured the rate of extracellular diffusion by injecting GdDOTA alone into S1 (n = 4). GdDOTA injections immediately produced a strong signal enhancement throughout a large region of the cortex, as one would expect

from rapid extracellular diffusion (Figure S4B, blue line in S4C). Moreover, the enhancement due to GdDOTA alone cleared extremely rapidly: it peaked at the first data point, immediately after injection, and it cleared completely within 24 hr. Thus, the GdDOTA was completely cleared well before the enhancement due to neuronal transport (i.e., from the GdDOTA-CTB) peaked (days 5–7, cf. red versus blue lines in Figure S4C). buy CP-673451 An additional control experiment was designed to further rule out the possibility that GdDOTA-CTB transport can be mediated by nonselective, passive uptake and diffusion. To test that hypothesis,

we injected another contrast compound, Gd-Albumin, in which the gadolinium was conjugated with bovine serum albumin (a protein that has a molecular weight similar to CTB). Although the injected gadolinium concentration Carnitine dehydrogenase from Gd-Albumin was comparable to the GdDOTA-CTB (i.e., 65–75 mM), and the protein concentration from Gd-Albumin was also very high (30% protein), we found that the signal intensity at the

Gd-Albumin injection sites was much weaker (compare Figure S4, red line, and Figure S5, solid black line), and the enhancement was much smaller, indicating that Gd-Albumin was rapidly cleared immediately after injection. The signal intensity at the injection core continued to decrease on day 4, returning to baseline values by day 7 (Figure S5). Except for the focal injection cores, no enhancement was present anywhere in the brain, including the thalamus, at any MR imaging time point. Overall, these data further support the conclusion that the GdDOTA-CTB conjugate is actively and selectively taken up and transported within the brain, as a MR-visible anatomical tracer. To clarify the relative advantages or disadvantages of GdDOTA-CTB, we compared GdDOTA-CTB results with comparable data using the tract-tracing contrast agent manganese. For both tracers, the signal intensity was measured at both the S1 injection site and the thalamic transport site, at comparable time points across tracers (i.e., immediately postinjection, at and after peak transport times for the two tracers). The two contrast agents were injected at the same concentration and volume.