To our knowledge, this study is the first to demonstrate brain connections in vivo, using a purpose-designed compound combining a classic neuroanatomical tracer (here, cholera-toxin subunit-B, CTB) with a known MRI-visible label (gadolinium-chelate, GdDOTA). When injected into S1 of rats, this compound produced a distinct enhancement of MR signal in multiple known thalamic targets of S1, including VPL, Rt, Po, and VM, in the hemisphere ipsilateral to the injection site (see , ). Moreover, the presumptive transport was somatotopically specific: injections aimed in the forepaw representation of S1 produced MR enhancement in the middle subfield of VPL, which corresponds to the forepaw representation in that thalamic nucleus (see , , , , middle panel).
This MR evidence for neural transport was confirmed by histology. Histological staining showed definitive CTB transport in the same thalamic nuclei that showed enhancement in the MR images, within the same animals, in the expected cellular compartments. For example, cell bodies and terminals were labeled in VPL, whereas only terminals were labeled in Rt ().
Outside the thalamus, the GdDOTA-CTB also showed additional MRI properties consistent with those known from classic tracers. This evidence included stable and long-lasting enhancement of MRI at the injection site, laminar-specific intrinsic connections near the injection site, connections with ipsilateral striatum and M1, and white matter projections beneath the injection site.
Time course of transport
Crucially, the time course of the thalamic MR enhancement is consistent with the interpretation of axonal transport of the GdDOTA-CTB compound. That MR enhancement began in the thalamic targets only after 2–3 days, and the enhancement peaked from 1–4 weeks post-injection (see ).
To the extent that it is known, histological evidence on CTB transport matches the time course of the presumptive transport of GdDOTA-CTB into thalamus, based on MRI. For axonal distances comparable to those in this study, CTB transport can first be detected 3–4 days following injection, and 7–14 days yield optimal results (Bruce and Grofova, 1992
; Erison and Blomovist, 1988
; Angulucci et al., 1996
; Sakai et al., 1998
). This similarity in time courses strongly supports our hypothesis that the MR signal enhancement in thalamus reflects active neuronal transport of GdDOTA-CTB to/from S1.
By comparison, MR enhancement due to passive extracellular diffusion (from GdDOTA injections into S1) peaked and then cleared within a day (see Fig. S4B–C
) – i.e., 4 days before the thalamic MR enhancement due to presumptive transport from GdDOTA-CTB reached statistical threshold. Moreover, the extracellular diffusion (GdDOTA alone) spread quite widely, unlike the specific target(s) enhanced following GdDOTA-CTB injections. Thus, the GdDOTA-CTB results were quite distinct from those due to extracellular diffusion, both temporally and spatially.
Although the GdDOTA-CTB showed strong and stable MR enhancement for long periods of time (at both the injection site and the targets), injections at similar concentrations of the control contrast compound, Gd-Albumin, cleared rapidly at the injection site - despite having a similar molecular weight. This suggests that local astrocytes and neurons take up Gd-Albumin non-specifically. Moreover, the lack of transport of the Gd-Albumin here and previously (Nagaraja et al., 2006
; Astary et al., 2010
) indicates that the GdDOTA-CTB works successfully as a unique MRI-visible tract-tracer, based on active uptake and transport processes.
Optimizing MRI enhancement
Using conventional T1-W MR sequences, GdDOTA-CTB produced a thalamic enhancement of 10–20% above the background MR level. A more targeted background-suppression T1-IR MR sequence yielded much higher signal increases (~ 80%). However the exact level of statistical sensitivity of this technique will vary widely depending on multiple technical factors. For instance, increases in the number of scans will increase the SNR ratio, in accord with the well-known inverse square law of signal averaging (I = 1/d2). Difference imaging (e.g. ) will also increase the statistical sensitivity. Difference imaging has been crucial in the fields of fMRI and optical recording, which are routinely based on significant signal variations as low as 0.1%. Thus, the current GdDOTA-CTB procedure produces signal changes that are well above the limits of statistical uncertainty.
Another crucial factor is the tracer molecule itself. For instance, the optimal ratio of Gd to CTB is not known. Results here were achieved with a ratio of 1.3–3 Gd/protein. However in a separate batch with up to 5 Gd per CTB (not described here), transport was not detected. Thus there may be an upper limit to the number of Gd that can be chelated and still yield effective CTB transport. Presumably, ratios that are too low sacrifice MRI sensitivity, whereas ratios that are too high may compromise uptake and/or transport.
The level of MR enhancement will also vary with the density of Gd reaching the target, which in turn reflects the divergence or convergence of those neural connections. Here our injections were concentrated in ~ 3–4 mm3
of S1 cortex. S1 projections converge onto, and arise from, much smaller (~ 1 mm3
) thalamic targets in VPL and Po; other thalamic targets are even smaller. Thus the convergence of these connections may concentrate Gd levels in thalamus. Anatomical studies support this idea: it has been reported that connections to/from S1 are more abundant with the thalamus (up to 1:40), compared with cortical targets (Sherman and Koch, 1990
). This factor may partially explain why cortico-cortical connections (e.g., from S1 to ipsilateral S2) were not apparent in our experiments, because cortical-cortical connections do not show such convergence. Technical limitations due to coil size and placement also reduced the detection of MR enhancement in S2 (see Supplemental Information
Toxicity and tissue damage after intra-cortical injections
Inevitably, insertion of an injection needle into the brain produces tissue damage along the needle track at the site of injection; it can also cause a small necrotic zone at the center of the needle tip (Fig. S1
). The relationship between transport and such tissue damage has a long and complex history in the literature on classical tracers. For example, it has been reported that injections of CTB in large volumes, or rapidly by pressure, can produce damage at the injection site (Conte et al., 2009a
). However, such tissue disruption is comparable following injections of the same amount of saline. Therefore, the important issue is whether damage occurs in the transport zones. Based on saline injection controls (Fig. S1
) and histology, we found no evidence of damaged cells in the transport zones. Even after long survival times post-injection, we did not observe decays in MR enhancement, thus ruling out the possibility of secondary degeneration in these remote transport zones.
Anterograde vs. retrograde transport
Histological studies have shown that CTB is transported in both directions along axons, both retrogradely to cell bodies, and anterogradely to presynaptic terminals (Luppi et al., 1986
; Bruce and Grofova, 1992
; Angeucci et al., 1996
; Sakai et al., 1998
; Wu and Kaas, 2000
). Is our CTB-based compound also transported in both directions? The current results clarify half of this two-part question. As is typical in the brain, transport from S1 to VPL, and from S1 and Po, are known to be reciprocal. Thus in these areas, our MR enhancement does not distinguish between the two directions of transport. However connections between S1 and Rt and CPu are atypically unidirectional: S1 projects to both Rt and CPu, but neither Rt nor CPu projects back to S1 (Kaas and Ebner, 1998
; Liu and Jones, 1999
. Gerfen, 1989
; Kincaid and Wilson, 1996
; Hoover et al., 2003
). In addition, it is known that sensory neurons in the olfactory epithelium project anterogradely to the OB. Thus we can conclude that the GdDOTA-CTB is transported anterogradely, at least.
Other techniques of tracing connections: Manganese
Injections of manganese chloride, coupled with MR imaging (MEMRI), have also been widely used to map brain connections in vivo
(Pautler et al., 1998
; Saleem et al., 2002
; Wu et al., 2006
; Tucciarone et al., 2009
; Chuang and Koretsky, 2009
). However, it is complicated to interpret the relationship of MEMRI data to the density of anatomical connections. First, MRI enhancement due to manganese reflects functional (i.e. calcium-related) activity (Lin et al., 1997
, Aoki et al., 2002
; Yu et al., 2005
; Eschenko et al., 2010a
) as well as anatomical connections. Secondly, manganese may be released from tissue after uptake (unpublished observations); manganese at the injection sites spreads quickly and continuously (e.g. and S6
, see also Tucciarone and Koretsky, 2009
). Thus MEMRI has not been used to measure connections across very small distances, such as those across cortical laminae. Thirdly, manganese is transported multisynaptically, not monosynaptically. In some experiments, this multisynaptic transport can be an advantage. However as described above, this can be a disadvantage if it is crucial to define each serial step of a given circuit. Moreover, diffusion of the manganese, coupled with the multisynaptic transport, could produce non-specific transport.
For these reasons, it can be challenging to distinguish which factor produces the observed MR enhancement in MEMRI studies. Additional measurements may be necessary. For instance, results from MEMRI have been compared to those from a classical tracer, to distinguish activity-dependent transport of manganese from anatomically based transport (Wu et al., 2006
; Saleem et al., 2002
The GdDOTA-CTB technique does not have these problems. Moreover, apparent disadvantages of the GdDOTA-CTB may be resolved by slight changes in procedure. For instance, multisynaptic connections could still be resolved using the monosynaptic transport of GdDOTA-CTB, using serial injections. For example, injections of GdDOTA-CTB into site A would produce transport to site B. Then a later MR-targeted injection into site B would produce transport to site C, and so on.
Other techniques of tracing connections: Iron oxide
Previous studies (Enochs et al. 1993
, van Everdingen et al. 1994
) reported slow transport (~ 5 mm/day) of dextran-coated iron oxide compounds, which were visible using MRI. However, that compound was specifically not transported in the central nervous system (CNS), when injected into either the superior colliculus or the eye (Enochs et al., 1993
). Prior to our use of GdDOTA-CTB, we also tested for CNS transport using an iron-labeled compound (biocytin conjugated with iron oxide). Consistent with the above findings, we also found that the biocytin-iron oxide compound did not produce transport, perhaps because iron-based compounds are too heavy to be transported easily in the CNS.
Other techniques of tracing connections: Classical tracers
The in vivo MRI-based tracer approach reveals connections that would be difficult or impossible to study otherwise. However, current MRI tracers will not supplant classical tracers (e.g. HRP, CTB, WGAHRP, etc.) because the latter can distinguish labeled cells from labeled presynaptic terminals, and thus reveal retro- vs. anterograde transport. Accordingly, classical tracers remain the gold standard, when such tracers are compatible with the experimental goals.
Other techniques of tracing connections: DTI and functional connections
Recently, invasive studies in animals have demonstrated functional connections more directly, by combining fMRI with electrical microstimulation of a targeted neural site (Tolias et al., 2005
; Ekstrom et al., 2008
; Moeller et al., 2008
; Field et al., 2008
). Although this technique raises exciting new possibilities, it has its own limitations. Anatomical connections can only be inferred, because the white matter pathways are not revealed. Moreover, the technique does not distinguish monosynaptic connections from multisynaptic ones. By comparison, the interpretation of anatomical connections using GdDOTA-CTB is simpler; it has all the properties of a classic neural tracer, without requiring animal sacrifice.
Presumably, future applications of the GdDOTA-CTB approach (perhaps in combination with other MRI-based techniques, such as DTI, resting state fMRI, and/or MEMRI) will furnish a much richer in vivo
diagram of brain circuitry. At a practical level, this MR-visible tracer can be used to target anatomically connected regions, using electrophysiological microelectrodes and/or serial tracer injections. More generally, this in vivo
approach can reveal changes occurring during plasticity induced by normal or abnormal physiology (e.g. axonal pruning or sprouting), in both central and peripheral nervous systems (Rakic et al., 1986
; LaMantia and Rakic, 1990
; Wu and Kaas, 2000
; Raff et al., 2002
; Madana and Esiri, 2003
; Prince et al., 2009