Infralimbic prefrontal cortex structural and functional connectivity with the limbic forebrain: a combined viral genetic and optogenetic analysis


The medial prefrontal cortex is critical for contextual appraisal, executive function, and goal-directed behavior. Additionally, the infralimbic (IL) subregion of the prefrontal cortex has been implicated in stress responding, mood, and fear memory. However, the specific circuit mechanisms that mediate these effects are largely unknown. To date, IL output to the limbic forebrain has been examined largely qualitatively or within a restricted number of sites. To quantify IL presynaptic input to structures throughout the forebrain, we utilized a lentiviral construct expressing synaptophysin-mCherry. Thus, allowing quantification of IL efferents that are specifically synaptic, as opposed to fibers of passage. Additionally, this approach permitted the determination of IL innervation on a sub-structural level within the multiple heterogeneous limbic nuclei. To examine the functional output of the IL, optogenetic activation of IL projections was followed by quantification of neuronal activation throughout the limbic forebrain via fos-related antigen (Fra). Quantification of synaptophysin-mCherry indicated that the IL provides robust synaptic input to a number of regions within the thalamus, hypothalamus, amygdala, and bed nucleus of the stria terminalis, with limited input to the hippocampus and nucleus accumbens. Furthermore, there was high concordance between structural connectivity and functional activation. Interestingly, some regions receiving substantial synaptic input did not exhibit significant increases in Fra-immunoreactivity. Collectively, these studies represent a step toward a comprehensive and quantitative analysis of output circuits. This large-scale efferent quantification or ‘projectome’ also opens the door for data-driven analyses of the downstream synaptic mechanisms that mediate the integrative aspects of cortico–limbic interactions.

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Third ventricle


Alexa Fluor 488


Anterior BST


Adeno-associated virus


Anterior hypothalamic nucleus


Anteroventral thalamus


Brodmann area 25


Basolateral amygdala


Basomedial amygdala


Bed nucleus of the stria terminalis


Anterolateral BST


Dorsal portion of anterolateral BST


Ventral portion of anterolateral BST


Anteromedial BST


Dorsal portion of anteromedial BST


Ventral portion of anteromedial BST


Fusiform BST


Interfascicular BST


Oval BST


Principal BST


Transverse BST


Cornu ammonis field 1


Cornu ammonis field 3


Ca2+/calmodulin-dependent protein kinase II alpha


Corpus callosum


Central nucleus of the amygdala


Lateral subdivision of central amygdala


Medial subdivision of central amygdala




Centromedial thalamus


Cortical amygdala




Dentate gyrus


Dorsomedial hypothalamus


Corpus callosum anterior forceps




Fos-related antigen


Glutamic acid decarboxylase, 67 kDa isoform




Infralimbic cortex


Lateral amygdala


Lateral hypothalamus


Lateral habenula




Medialdorsal thalamus


Major depressive disorder


Medial amygdala


Anterodorsal MeA


Posterodorsal MeA


Posteroventral MeA


Manders’ overlap coefficient


Medial prefrontal cortex


Medial preoptic nucleus


Medial preoptic area


Mammillothalamic tract


Nucleus accumbens


NAc core


NAc shell


Neuronal nuclear protein


Optic tract


Posterior BST


Posterior hypothalamic nucleus


Prelimbic cortex


Paratenial thalamus


Paraventricular nucleus of the hypothalamus


Paraventricular thalamus


Nucleus reunions




Stria medullaris


Stria terminalis


Ventromedial hypothalamus


Ventral subiculum


Yellow fluorescent protein


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AAV vectors were provided by the University of North Carolina Vector Core under material transfer agreement with Karl Deisseroth and Stanford University.


This work was supported by NIH grant K99/R00 HL122454 and an American Heart Association Fellowship to B. Myers, as well as NIH Grants R01 MH049698 and R01 MH101729 to J. P. Herman. T. Wallace received support from the Colorado State University Molecular, Cellular and Integrative Neuroscience program.

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Correspondence to Brent Myers.

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All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee and comply with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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Wood, M., Adil, O., Wallace, T. et al. Infralimbic prefrontal cortex structural and functional connectivity with the limbic forebrain: a combined viral genetic and optogenetic analysis. Brain Struct Funct 224, 73–97 (2019).

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  • Anterograde
  • Fos-related antigen
  • Rat
  • Synaptophysin