Abstract
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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Abbreviations
- 3v:
-
Third ventricle
- A488:
-
Alexa Fluor 488
- aBST:
-
Anterior BST
- AAV:
-
Adeno-associated virus
- AHN:
-
Anterior hypothalamic nucleus
- AV:
-
Anteroventral thalamus
- BA25:
-
Brodmann area 25
- BLA:
-
Basolateral amygdala
- BMA:
-
Basomedial amygdala
- BST:
-
Bed nucleus of the stria terminalis
- BSTal:
-
Anterolateral BST
- BSTald:
-
Dorsal portion of anterolateral BST
- BSTalv:
-
Ventral portion of anterolateral BST
- BSTam:
-
Anteromedial BST
- BSTamd:
-
Dorsal portion of anteromedial BST
- BSTamv:
-
Ventral portion of anteromedial BST
- BSTfu:
-
Fusiform BST
- BSTif:
-
Interfascicular BST
- BSTov:
-
Oval BST
- BSTpr:
-
Principal BST
- BSTtr:
-
Transverse BST
- CA1:
-
Cornu ammonis field 1
- CA3:
-
Cornu ammonis field 3
- CaMKIIα:
-
Ca2+/calmodulin-dependent protein kinase II alpha
- cc:
-
Corpus callosum
- CeA:
-
Central nucleus of the amygdala
- CeL:
-
Lateral subdivision of central amygdala
- CeM:
-
Medial subdivision of central amygdala
- ChR2:
-
Channelrhodopsin-2
- CM:
-
Centromedial thalamus
- CoA:
-
Cortical amygdala
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DG:
-
Dentate gyrus
- DMH:
-
Dorsomedial hypothalamus
- fa:
-
Corpus callosum anterior forceps
- fx:
-
Fornix
- Fra:
-
Fos-related antigen
- GAD67:
-
Glutamic acid decarboxylase, 67 kDa isoform
- IHC:
-
Immunohistochemistry
- IL:
-
Infralimbic cortex
- LA:
-
Lateral amygdala
- LH:
-
Lateral hypothalamus
- LHab:
-
Lateral habenula
- mCh:
-
mCherry
- MD:
-
Medialdorsal thalamus
- MDD:
-
Major depressive disorder
- MeA:
-
Medial amygdala
- MeAad:
-
Anterodorsal MeA
- MeApd:
-
Posterodorsal MeA
- MeApv:
-
Posteroventral MeA
- MOC:
-
Manders’ overlap coefficient
- mPFC:
-
Medial prefrontal cortex
- MPN:
-
Medial preoptic nucleus
- mPOA:
-
Medial preoptic area
- mtt:
-
Mammillothalamic tract
- NAc:
-
Nucleus accumbens
- NAcC:
-
NAc core
- NAcS:
-
NAc shell
- NeuN:
-
Neuronal nuclear protein
- ot:
-
Optic tract
- pBST:
-
Posterior BST
- PH:
-
Posterior hypothalamic nucleus
- PL:
-
Prelimbic cortex
- PT:
-
Paratenial thalamus
- PVN:
-
Paraventricular nucleus of the hypothalamus
- PVT:
-
Paraventricular thalamus
- Re:
-
Nucleus reunions
- Sph:
-
Synaptophysin
- sm:
-
Stria medullaris
- st:
-
Stria terminalis
- VMH:
-
Ventromedial hypothalamus
- vSub:
-
Ventral subiculum
- YFP:
-
Yellow fluorescent protein
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Acknowledgements
AAV vectors were provided by the University of North Carolina Vector Core under material transfer agreement with Karl Deisseroth and Stanford University.
Funding
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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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). https://doi.org/10.1007/s00429-018-1762-6
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DOI: https://doi.org/10.1007/s00429-018-1762-6