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. Tract tracing after MnCl2 stereotactic intracerebral injection is performed by time-lapse MRI in living mice, based on harnessing the intracellular axonal transport machinery, kinesin and microtubules. Volumes of statistically significant, Mn(II)-dependent, image enhancements report on distal accumulations brain wide that can be measured segment-wise for statistical comparisons of the relative strength of the projection contacts. Targets of neuronal projections from the medial prefrontal cortex differ between anterior cingulate (ACA, red) and infralimbic-prelimbic (IL/PL, blue) areas. While neurons in ACA project to central amygdala, posterior hypothalamic regions, periaqueductal gray, dorsal raphe, and locus coeruleus; those in IL/PL project to basolateral amygdala, anterior hypothalamic subsegments, central superior raphe and pons.

Automated summary

Neurons in the medial prefrontal cortex project into the limbic system to regulate responses to reward or threat, with different regions projecting to differing limbic system nuclei. Using Mn(II) and MRI, projections from two adjacent medial prefrontal cortical regions were mapped, revealing distinct accumulation patterns in diverse brain regions over time. This study sheds light on the specific roles of different medial prefrontal cortical segments in regulating limbic system function and provides insights into the potential effects on whole body health and wellbeing.