Harnessing axonal transport to map reward circuitry: Differing brain-wide projections from medial prefrontal cortical domains

Neurons project long axons that contact other distant neurons. Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat. Diminished neural activity in prefrontal cortex is associated with loss of executive function leading to drug use, yet the specific circuitry that mediate these effects is unknown. Different regions within the medial prefrontal cortex may project to differing limbic system nuclei. Here, we exploited the cell biology of intracellular membrane trafficking, fast axonal transport, to map projections from two adjacent medial prefrontal cortical regions. We used Mn(II), a calcium analog, to trace medial prefrontal cortical projections in the living animal by magnetic resonance imaging (MRI). Mn(II), a contrast agent for MRI, enters neurons through voltage-activated calcium channels and relies on kinesin-1 and amyloid-precursor protein to transport out axons to distal destinations. Aqueous MnCl2 together with fluorescent dextran (3–-5 nL) was stereotactically injected precisely into two adjacent regions of the medial prefrontal cortex: anterior cingulate area (ACA) or infralimbic/prelimbic (IL/PL) region. Projections were traced, first live by manganese-enhanced MRI (MEMRI) at four time points in 3D, and then after fixation by microscopy. Data-driven unbiased voxel-wise statistical maps of aligned normalized MR images after either ACA or IL/PL injections revealed statistically significant progression of Mn(II) over time into deeper brain regions: dorsal striatum, globus pallidus, amygdala, hypothalamus, substantia nigra, dorsal raphe and locus coeruleus. Quantitative comparisons of these distal accumulations at 24 h revealed dramatic differences between ACA and IL/PL injection groups throughout the limbic system, and most particularly in subdomains of the hypothalamus. ACA projections targeted dorsomedial nucleus of the hypothalamus, posterior part of the periventricular region and mammillary body nuclei as well as periaqueductal gray, while IL/PL projections accumulated in anterior hypothalamic areas and lateral hypothalamic nuclei as well as amygdala. As hypothalamic subsegments relay CNS activity to the body, our results suggest new concepts about mind-body relationships and specific roles of distinct yet adjacent medial prefrontal cortical segments. Our MR imaging strategy, when applied to follow other cell biological processes in the living organism, will undoubtedly lead to an expanded perspective on how minute details of cellular processes influence whole body health and wellbeing.


Supplementary Table
. Abbreviations used in the column graphs in Fig. 6 and 8, in the order in which the columns appear in the graph.Nomenclature is according to the Allen Institute for Brain Science Mouse Brain Reference Atlas.Shown are three coronal slices showing regions with statistically significant accumulations (T = 5.56, p<0.01 FDR) for all projections.Color coding is the same as for Figure 6 in the main text, red, 6h>30m; blue, 24h>6h; and turquoise, 24h > 6h.Slice positions shown in the corner of each image.
Top panel shows accumulation in the Globus pallidus (GPe) that progresses from 6h (blue) to 24h (red), with greater accumulation on the injection side (R).Mouse is facing forwards.
Middle panel shows statistically significant intensity increases due to Mn(II) accumulations differ between time points.Significantly, the Parafascicular nucleus of the thalamus accumulates signal first, appearing in the 6h>30m (red) map (Parafascicular nucleus, PF).Note that the Mediodorsal thalamic nucleus (MD), a known direct projection from the motor cortex (MO) is not highlighted at any time point.Signal also appears in this 6h map in a region running below the Zona incerta (ZI, purple outline).The ZI itself, another direct target of MO, has no signal at any time point.In scrolling through slices, signal in the cpd can be followed into the substantia nigra reticulata (SNr), where signal is robust at all timepoints.This trajectory below the ZI may represent the cerebral peduncle (cpd), which we purposefully had omitted from our segmentation analysis as cpd is a fiber bundle as we expected ACA projections would be diffuse and not appear in fiber bundles.However, others have recently reported transport in cerebral peduncle after ACA injection of a viral histologic tracer (Shi et al 2021)..At 24h (dark blue), signal in the cpd continues to be present and also newly appears in the retrosplenial area (RSP).The lack of signal in the cortex at 6h (no red) suggests that signal in the retrospenial area is not from Mn(II) diffusion out of the injection site, but rather transport from some other area, either through direct or trans-synaptic connections.
Signal appears only in comparisons between 6h and 24h (turquoise) in some regions, which may indicate slower arrival of the Mn(II), even in those that are closer as the crow flies to the injection site than the SNr.These late appearing accumulations appear in hippocampus (CA1 and part of the dentate on the injected side (R), and some in the CA1 field on the contralateral (L) side,) as well as in retrosplenial (RSP) and motor cortex (MO).
Lower panel shows statistically significant accumulation in substantia nigra reticulata (SNr) at 6hr (red) and 24h (dark blue).In cortex in this same slice, it can also be seen that signal arrives at the cortex at 24h.

Table S2 .
Coordinates for Regions of Interest Analysis relative to Bregma (in mm)

Table S3 . ROI Measurements See Supplementary Table S3, a separate file available as Excel. Supplementary Table S4. ROI Statistics (pdf)
Supplementary Table S4.Statistics for ROI within and between group analyses Figures 5 and 7 in the Main text.